Which of Perutz et al's protein structures other than 3₁₀ helix were correct?

Which of Perutz et al's protein structures other than 3₁₀ helix were correct?

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In 1950, Bragg, Kendrew and Perutz published "Polypeptide chain configurations in crystalline proteins" (open access) and were famously 'proved wrong' by Pauling, Corey and Branson the following year, in the paper that documented the alpha and gamma helices, "Two Hydrogen-Bonded Helical Configurations of the Polypeptide Chain" (also OA)

I'm reading about this (as reviewed here for example) where the idea is that Bragg et al were disproved - in the words of the Pauling paper:

None of these authors propose either our 3.7-residue [α] helix or our 5.1- residue [γ] helix. On the other hand, we would eliminate by our basic postulates all of the structures proposed by them. The reason for the difference in results obtained by other investigators and by us through essentially similar arguments is that both Bragg and his collaborators… discussed in detail only helical structures with an integral number of residues per turn, and moreover assume only a rough approximation to the requirements about interatomic distances bond angles, and planarity of the conjugated amide group, as given by our investigations of simpler substances. We contend that these stereochemical features must be very closely retained in stable configurations of polypeptide chains in proteins, and that there is no special stability associated with an integral number of residues per turn in the helical molecule.

There was one however, the 310 helix which was correctly identified by Perutz's group in 1950. I don't have any access to library facilities at present and online resources aren't quite the same as a solid textbook. I'm wondering if any of the other forms described were in fact correct?

To list, these were:

  • 27a
  • 27b
  • 28
  • 213
  • 214
  • 38
  • ( 310 )
  • 411
  • 413
  • 213

I'll continue looking in the meantime but it's hard to search when there are subscripts in names, and I would think more experienced protein scientists could give me a quicker answer (or just direct me to where I should be reading)

I think you've got the list of good predictions from the Bragg, Perutz, and Kendrick paper. And the 310 helix was not really right either - it did turn out to show up occasionally in protein structures though.

At the time all of these secondary structure elements were well evidenced from noncrystalline diffraction data and small molecule crystal structures. Examples being collagen from hair and spider silk fiber diffraction patterns. Fiber diffraction of hair, whose ordered structure consists mainly of alpha helices somewhat aligned to the fiber axis (along the length of the hair) or the mainly beta sheets which constitute silk.

Because it was known that these substances contain mainly protein, building models which would show how regular peptide polymers would produce these patterns. This happened in chemistry and physics departments worldwide. The paper you cite is representative to the field.

Pauling caused a splash in the spring of 1951 by publishing descriptions of the alpha helix and the beta sheet (both parallel and antiparallel). The main advantage that Pauling had was that he had studied the structure of simple di-amino acid structures (notably glycyl-glycine) and understood that the peptide bond had some double bond like properties which prevented it from rotating freely. As the discoverer of valence bond theory, which united quantum mechanics and chemistry and established the basis for at least most organic/biological chemical structures Pauling must have been practically alone in this insight at the time.

This next set the stage for the dramatic race to discover the structure of DNA. If you read Watsons' memoir, he describes how the MRC in Cambridge was still feeling the heat, competing with CalTech and Pauling. He and the young lab there was holding their own against the MRC Cambridge with its multiple nobel laureates; a lab which had established the relationship of x-ray diffraction and chemical structure (the notes on chapter 7 are interesting - follow the link).

Looking back on it, Pauling's main advantage was that he understood the peptide bond better than the rest of the world. By the time DNA was being resolved, the conformations of organic molecules were understood by many and the playing field should have been more level. I'm not sure I buy the arguments that Pauling would have got the structure without seeing the diffraction patterns and even then maybe not. I might not give such strong credence to the idea that Pauling would have gotten the DNA structure so quickly if he had been able to travel.

Of course Perutz and Kendrew verified the peptide alpha helix themselves when they solved the first crystal structures of hemoglobin and myoglobin, having the final say. The structures contain a small amount of 310 helix but are otherwise full of alpha helices. I'm not sure that the secondary structure was entirely confirmed until the 1958 when myoglobin was resolved.

An analysis of incorrectly folded protein models : Implications for structure predictions☆

Proteins with homologous amino acid sequences have similar folds and it has been assumed that an unknown three-dimensional structure can be obtained from a known homologous structure by substituting new side-chains into the polypeptide chain backbone, followed by relatively small adjustment of the model. To examine this approach of structure prediction and, more generally, to isolate the characteristics of native proteins, we constructed two incorrectly folded protein models. Sea-worm hemerythrin and the variable domain of mouse immunoglobulin κ-chain, two proteins with no sequence homology, were chosen for study the former is composed of a bundle of four α-helices and the latter consists of two 4-stranded β-sheets. Using an automatic computer procedure, hemerythrin sidechains were substituted into the immunoglobulin domain and vice versa. The structures were energy-minimized with the program CHARMM and the resulting structures compared with the correctly folded forms. It was found that the incorrect side-chains can be incorporated readily into both types of structures (α-helices, β-sheets) with only small structural adjustments. After constrained energy-minimisation, which led to an average atomic co-ordinate shift of no more than 0.7 to 0.9 Å, the incorrectly folded models arrived at potential energy values comparable to those of the correct structures. Detailed analysis of the energy results shows that the incorrect structures have less stabilizing electrostatic, van der Waals' and hydrogen-bonding interactions. The difference is particularly pronounced when the electrostatic and van der Waals' energy terms are calculated by modified equations that include an approximate representation of solvent effects. The incorrectly folded structures also have a significantly larger solvent-accessible surface and a greater fraction of non-polar side-chain atoms exposed to solvent. Examination of their interior shows that the packing of sidechains at the secondary structure interfaces, although corresponding to sterically allowed conformations, deviates from the characteristics found in normal proteins. The analysis of incorrectly folded structures has made it clear that the absence of bad non-bonded contacts, though necessary, is not sufficient to demonstrate the validity of model-built structures and that modeling of homologous structures has to be accompanied by a thorough quantitative evaluation of the results. Further, certain features that characterize native proteins are made evident by their absence in misfolded models.

Dr. Pauling’s Chiral Aliens

[A guest post expanding on Pauling’s idea for a science fiction novel. Post authored by the blog’s East Coast Bureau Chief, Dr. John Leavitt, Nerac, Inc., Tolland, CT.]

Pauling lecturing with the “fish model” (foreground) that he used to demonstrate chirality, ca. 1960s.

In basic chemistry we have something called “chirality” which refers to a molecule with two possible non-superimposable configurations. One way to picture this is to look at your hands and place one on top of the other (not palm to palm) – your left and right hands are essentially the same shape but their shape is reversed. At the molecular level we can use one of the main building blocks of all proteins and all life – the amino acid alanine, depicted in the image below – to examine handedness.

The diagram shows the arrangement of atoms of two alanine molecules, both of which exist in nature, arranged so that they are mirror images. They are the same molecules but if you turn the one on the right around so that it is facing in the same direction as the one on the left, the R (a single carbon atom in alanine with three bonded hydrogen atoms) on this alanine molecule faces toward the palm of the hand and the COOH moiety (a carboxyl group) and the NH2 moiety (an amino group) face outward away from the palm.

No matter how you rotate the alanine on the right, you can’t get the three moieties attached to the central carbon to line up in the same position as the alanine on the left. Likewise, you can’t get those hands to super-impose each other no matter how much you twist and turn them. So the alanine on the left is called L-alanine (levo- for the direction the molecule rotates photons) and the alanine on the right is called D-alanine (dextro- for the direction the molecule rotates photons). They are called “enantiomers,” or chiral forms, of alanine, and both exist in nature with identical chemical properties except for the way that they rotate polarized light.

There are twenty natural amino acids comprising the building blocks of all proteins. Of these twenty, only glycine is symmetrical around a central carbon atom and therefore glycine has no enantiomers. The other nineteen can exist in the L- and D-conformation.

Funny thing though, only the L-enantiomer is used to make proteins by the protein synthetic machinery of all life-forms, from single-cell organisms up to humans. It’s quite easy to understand why one enantiomer is used in life over random use of either enantiomer. In explaining this, note the pictures below, which show the three-dimensional globular structure of human beta-actin on the left and, on the right, the architectural arrangement of this actin in the cytoplasm of a cell.

The protein composed of 374 amino acids has an intricate folding pattern with coils which would not be possible if both amino acid enantiomers for the nineteen amino acids were randomly incorporated into the protein. This three-dimensional structure has to be preserved in order for actin to perform its dynamic architectural function inside living cells, as shown in the picture on the right. The coils are possible because the amino acids are all L-amino acids and glycine is neutral otherwise the protein would behave like a wet noodle. The precise structure of the actin protein determines its function, which has been preserved and conserved since the beginning of all eukaryotic life-forms (that is, cells with a cytoplasm and a nucleus). Understanding the atomic forces that fold proteins in a unique shape is part of the reason why Linus Pauling received the Nobel Prize for Chemistry in 1954.

Aside from those who closely follow this blog, it is not well known that Linus Pauling was an avid reader of science fiction. In a 1992 interview with biographer Thomas Hager, he described his motivation to write a science fiction novel. The story line was to be the discovery of a human-like race from another planet that had evolved to use only D-amino acids (D-humans) rather than the L-isoform (L-humans). He explained that he never got around to writing this novel because the real science he was doing took all of his time.

If our L-humans met up with those D-humans, what consequences would there be? Well, what we would see in D-humans are people virtually indistinguishable from ourselves – barring, of course, the possibility that these extraterrestrials evolved out of some unearthly environmental niche. However, no mating, blood, or tissue sharing would be possible between these two races.

To explain this, consider the experience you have had when you accidently put your hand in the wrong glove. As you know, this doesn’t work well. All protein interactions and reactions catalyzed by enzymes require a direct fit to work. Substrates of enzymes have to fit precisely into the catalytic active site of the enzyme, like your hand fitting into the correct glove. Since L-humans have a different chirality from D-humans, nothing would fit or be transferrable, because of asymmetric incompatibility between L- and D- macromolecules. Even the food on our planet would not likely be nutritious for D-humans because all living things on Earth are L-organisms. In D-lifeforms, the actin coils would coil in the opposite direction and the DNA double helix would have to spiral in the opposite direction as well otherwise the analogous D-proteins would not bind or fit on the chromosomal DNA.

It seems reasonable that D-humans might be found on other planets if you consider how life got started. By a quirk of nature on Earth, L-amino acids got a head start and self-assembled into peptides (small proteins) when this essential process for life as we know it got started. The assembly of only one enantiomer isoform into a peptide may have been favored thermodynamically over co-random assembly of L- and D-isoforms. This essential process evolved into a well-organized, membrane-protected and energy-driven protein synthetic machinery in single cell organisms like bacteria. Today, humans have essentially the same protein synthetic machinery that evolved in primordial bacteria and all life-forms on Earth have the same genetic code.

There are two essential enzymes that work together to catalyze protein synthesis in all living cells. One enzyme, called aminocacyl-tRNA synthetase, binds the amino acid to a transfer RNA molecule (there is one of these enzymes and a specific tRNA for each of the twenty amino acids). The second enzyme, peptidyl transferase, catalyzes the formation of a peptide bond linking two amino acids at the start of a chain and does this over and over again until the full length protein is synthesized and folded into its functional conformation. These two essential enzymes do not recognize the D-isoforms of the nineteen asymmetric amino acids. Thus, our chiral L-specificity has been preserved since the beginning of life.

I can’t think of any reason why the D-amino acids would not support life, but it has to be one isoform or the other, not both. Apparently Pauling felt the same way. Should it ever come to pass, D-humans will be interesting to meet and they will be equally interested to meet us, hopefully without mutual disappointment.

Location of helical regions in tetrapyrrole-containing proteins by a helical hydrophobic moment analysis. Application to phytochrome.

Helical regions in many tetrapyrrole proteins are highly amphiphilic, one side interacting with a hydrophobic core and another side interacting with the polar solvent. The mean helical hydrophobic moment is a measure of amphiphilicity of a helix. Helical regions in myoglobin, the alpha and beta subunits of C-phycocyanin, and cytochrome c can be distinguished from nonhelical regions by use of a hydrophobic moment analysis. 24 of 27 (89%) of the helical regions in these proteins were located by this analysis. Calculations were also performed on chymotrypsin, ribonuclease, and papain, which do not possess as pronounced a hydrophobic core as the tetrapyrrole-containing proteins. Less than 50% of the helical regions were correctly located, indicating a lack of amphiphilicity in the helices of these proteins. The hydrophobic moment analysis was also used to predict helical regions in phytochrome, the ubiquitous photoreceptor in plants. Additionally, this analysis is used to quickly locate internal hydrophilic residues which may be functionally important. The distribution of hydrophobic moments from a random sequence was determined so that qualitative and to some extent quantitative comparisons between different amphiphilic helices may be made.


We assembled Fourier maps with the aid of Beevers and Lipson strips, which were thin paper strips containing values of sines and cosines for given amplitudes and frequencies sampled at appropriate intervals, that we assembled and then summed to give the electron density projection. After a few years of this I completed the requirements for my D. Phil. and, thanks to Dunitz's suggestion, I went to Caltech to postdoc with Pauling and Corey. This was a mind-boggling experience on many counts. When we arrived in Pasadena, after flying low into the Grand Canyon, we stayed at the Athenaeum, the Caltech Faculty Club. The first morning, I opened the blinds and was astonished to see the 6000-foot facade of Mount Wilson. Dunitz had never mentioned this proximity of mountains almost twice as high as Snowdon, the tallest mountain in Wales. Then the smog descended and it was two months before we saw Mount Wilson again. To us, coming from an England that was just emerging from WWII rationing, California was a land overflowing with milk and honey. Then there was the weather, where months could pass without rain and seasonal differences were small. The Caltech social atmosphere was open and friendly, and there was a degree of socializing between faculty and students that would have been unthinkable at Oxford. In addition to the ocean we were within striking distance of the desert and the mountains, and many weekends were spent exploring the Sierra Nevada mountains and Death Valley.

However, the political climate in California was chillingly conservative. Richard Nixon had been elected vice president to Eisenhower shortly after we arrived. He was universally disliked by the postdocs and graduate students for the way he had campaigned against Jerry Voorhis and Helen Gahagan Douglas. However, the most striking manifestation of the general support for a reflex anticommunism lay in the phenomenon of Senator Joe McCarthy, whose rise and fall occurred during our stay in California. Pauling, who was outspoken on political matters, particularly pertaining to the control of nuclear weapons, was himself a victim of this atmosphere and was at one point denied a passport to visit a Royal Society meeting in London.

Caltech, however, was a wonderful place and we were most impressed with the caliber of the graduate students and postdocs, most of whom went on to distinguished careers in scientific research. I was partnered with Joe Blum, a physiologist who had come to Caltech to learn crystallography so that he could eventually work on the structure of muscle. There was still at Caltech an active program in crystallography aimed at accurate structures of small peptide-like molecules to improve the precision of the database that had been assembled for the construction of the α-helix and the β-sheets. I was asked to join this program, and Joe and I determined the structure of an eight-atom structure called parabanic acid. We collected a set of films containing the three-dimensional X-ray data, and we each independently measured the intensities visually, by comparison with an intensity scale that we constructed. The Caltech Chemistry Department was at the time probably the best place anywhere to practice crystallography. There was an array of IBM punched card computers that we could use, and the procedures for using them had been well established. Each machine could be “programmed” by wiring a plugboard, and they ranged from a simple sorter to the IBM 604, a computer that had a thousand vacuum tubes and could carry out sixty simple operations for the passage of each card. When we calculated a final difference map for the parabanic acid crystal, we found unexpected peaks that indicated that the molecule had anisotropic thermal vibration. So with the advice of John Rollett, who had come from Leeds, Joe and I developed a least squares procedure for refining the individual atomic anisotropic thermal vibration parameters using the three-dimensional data. This removed the unwanted peaks in the difference map, gave a suitably low R factor, and provided a remarkably precise set of atomic dimensions. In my second year I repeated this procedure with succinamide and was even able to see what looked like bonding electrons. With modern computers this procedure is now routinely applied to every crystal structure determination, but at the time, doing it for the first time was exciting (6, 7).

In the fall of 1953 there was a meeting at Caltech that was, I believe, originally intended to gather support for the Pauling and Corey structures. The α-helix model predicted a strong X-ray reflection close to the meridian at a spacing of 5.4 Å, whereas Astbury's fiber patterns showed a 5.1 Å reflection and this presented a problem for the structure that had caused Pauling to delay publication for some time. However, in the year preceding the conference there had been several extraordinary developments. First, Crick had provided a clever and plausible explanation for the difference between the observed and calculated spacings by proposing a coiled-coil structure that would result in a 5.1 Å reflection. Second, Max Perutz and his colleagues David Green and Vernon Ingram had just achieved the first successful isomorphous replacement in hemoglobin crystals using a mercury derivative that enabled them to calculate the phases for a projection of the molecule, thus demonstrating conclusively that the determination of protein structures was possible. Third, Watson and Crick had just determined the structure of DNA. These events combined to make this the most remarkable structural biology meeting I have ever attended and made even a committed small-molecule crystallographer appreciate the treasures that were to be discovered from a study of biological macromolecules.

However, it was not to be immediately and I returned to England to an industrial job in Oldbury, on the west of Birmingham on the edge of the “Black Country.” I worked in the research department of a company that manufactured phosphorus fine chemicals. It was my first introduction to industrial England, where the canal was green and greasy and the fogs impenetrable. However, the research department had a small but effective X-ray laboratory and I was able to determine the crystal structure of sodium triphosphate.

Dorothy Hodgkin, who put bio and crystallography together

Although Dorothy Crowfoot Hodgkin was not the first one to determine the crystal structure of a protein, her contribution to the field of macromolecular crystallography was truly monumental. She initially studied chemistry and then became a coworker of Bernal in Cambridge. Very quickly, she acquired excellent mastery of crystallography, buttressed by a first class knowledge of chemistry. She worked with Bernal on recording the first protein diffraction images of pepsin crystals [16] and, independently, already in Oxford, on obtaining diffraction photographs of insulin [21] . Insulin became her life-long interest, crowned eventually, after almost 35 years of effort, by solving the structure of this important protein hormone [22] . Although the molecule of insulin is not particularly large, solving the structure was complicated by the presence of two molecules in the asymmetric unit in space group R3. This space group lacks centric reflections, which were critical for solving the first crystal structures of hemoglobin and myoglobin (see below). Hodgkin continued structural studies of insulin until the end of her active scientific career, publishing what is most likely the longest paper in the history of protein crystallography, taking up a whole issue of Philosophical Transactions of the Royal Society of London, Series B [23] . The co-authors of this monumental work, listed in alphabetical order and all trained by Hodgkin, include such well-known structural biologists as Ted Baker, Tom Blundell, Eleanor and Guy Dodson, and Mamannamana Vijayan, amongst others. Guy Dodson (1937–2012), in particular, continued the studies of insulin for many more years, participating in the work that culminated in a key paper describing the structure of its complex with the insulin receptor [24] .

Even before her success with insulin, Dorothy Hodgkin was practically a biomacromolecular crystallographer because the structures successfully solved by her were not only difficult and large for that period (1930–1960), but also were extremely important from the chemical and biological points of view. Chemists at that time were not sure at all about the correct structure of sterols and several possible formulas were around with four aliphatic rings connected in various ways. The crystal structure of an iodo derivative of cholesterol unambiguously established the correct structure of sterols [25] . The crystal structure of penicillin, determined in the early 1940s, had a similarly enormous impact, surprising some chemists with the unexpected four-membered β-lactam ring. This achievement opened the route for making semisynthetic versions of this antibiotic, although it was not published until 1949 because of its military use at the end of World War II [26] . The crown jewel of Dorothy Hodgkin's work, for which she was awarded the 1964 Nobel Prize in Chemistry, was the structure of vitamin B12, the largest crystal structure solved at that time. It again revealed several unexpected features, such as the corrin ring structure and the covalent bond between cobalt and carbon atoms, making vitamin B12 the first identified organometallic compound. This work involved a pioneering application of the early electronic computers in a long-distance collaboration with Ken Trueblood (1920–1998) in California [27] .


Proteins were recognized as a distinct class of biological molecules in the eighteenth century by Antoine Fourcroy and others, distinguished by the molecules' ability to coagulate or flocculate under treatments with heat or acid. [1] Noted examples at the time included albumin from egg whites, blood serum albumin, fibrin, and wheat gluten.

Proteins were first described by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist Jöns Jacob Berzelius in 1838. [2] [3] Mulder carried out elemental analysis of common proteins and found that nearly all proteins had the same empirical formula, C400H620N100O120P1S1. [4] He came to the erroneous conclusion that they might be composed of a single type of (very large) molecule. The term "protein" to describe these molecules was proposed by Mulder's associate Berzelius protein is derived from the Greek word πρώτειος (proteios), meaning "primary", [5] "in the lead", or "standing in front", [6] + -in. Mulder went on to identify the products of protein degradation such as the amino acid leucine for which he found a (nearly correct) molecular weight of 131 Da. [4] Prior to "protein", other names were used, like "albumins" or "albuminous materials" (Eiweisskörper, in German). [7]

Early nutritional scientists such as the German Carl von Voit believed that protein was the most important nutrient for maintaining the structure of the body, because it was generally believed that "flesh makes flesh." [8] Karl Heinrich Ritthausen extended known protein forms with the identification of glutamic acid. At the Connecticut Agricultural Experiment Station a detailed review of the vegetable proteins was compiled by Thomas Burr Osborne. Working with Lafayette Mendel and applying Liebig's law of the minimum in feeding laboratory rats, the nutritionally essential amino acids were established. The work was continued and communicated by William Cumming Rose. The understanding of proteins as polypeptides came through the work of Franz Hofmeister and Hermann Emil Fischer in 1902. [9] [10] The central role of proteins as enzymes in living organisms was not fully appreciated until 1926, when James B. Sumner showed that the enzyme urease was in fact a protein. [11]

The difficulty in purifying proteins in large quantities made them very difficult for early protein biochemists to study. Hence, early studies focused on proteins that could be purified in large quantities, e.g., those of blood, egg white, various toxins, and digestive/metabolic enzymes obtained from slaughterhouses. In the 1950s, the Armour Hot Dog Co. purified 1 kg of pure bovine pancreatic ribonuclease A and made it freely available to scientists this gesture helped ribonuclease A become a major target for biochemical study for the following decades. [4]

Linus Pauling is credited with the successful prediction of regular protein secondary structures based on hydrogen bonding, an idea first put forth by William Astbury in 1933. [12] Later work by Walter Kauzmann on denaturation, [13] [14] based partly on previous studies by Kaj Linderstrøm-Lang, [15] contributed an understanding of protein folding and structure mediated by hydrophobic interactions.

The first protein to be sequenced was insulin, by Frederick Sanger, in 1949. Sanger correctly determined the amino acid sequence of insulin, thus conclusively demonstrating that proteins consisted of linear polymers of amino acids rather than branched chains, colloids, or cyclols. [16] He won the Nobel Prize for this achievement in 1958. [17]

The first protein structures to be solved were hemoglobin and myoglobin, by Max Perutz and Sir John Cowdery Kendrew, respectively, in 1958. [18] [19] As of 2017 [update] , the Protein Data Bank has over 126,060 atomic-resolution structures of proteins. [20] In more recent times, cryo-electron microscopy of large macromolecular assemblies [21] and computational protein structure prediction of small protein domains [22] are two methods approaching atomic resolution.

The number of proteins encoded in a genome roughly corresponds to the number of genes (although there may be a significant number of genes that encode RNA of protein, e.g. ribosomal RNAs). Viruses typically encode a few to a few hundred proteins, archaea and bacteria a few hundred to a few thousand, while eukaryotes typically encode a few thousand up to tens of thousands of proteins (see genome size for a list of examples).

Most proteins consist of linear polymers built from series of up to 20 different L-α- amino acids. All proteinogenic amino acids possess common structural features, including an α-carbon to which an amino group, a carboxyl group, and a variable side chain are bonded. Only proline differs from this basic structure as it contains an unusual ring to the N-end amine group, which forces the CO–NH amide moiety into a fixed conformation. [23] The side chains of the standard amino acids, detailed in the list of standard amino acids, have a great variety of chemical structures and properties it is the combined effect of all of the amino acid side chains in a protein that ultimately determines its three-dimensional structure and its chemical reactivity. [24] The amino acids in a polypeptide chain are linked by peptide bonds. Once linked in the protein chain, an individual amino acid is called a residue, and the linked series of carbon, nitrogen, and oxygen atoms are known as the main chain or protein backbone. [25] : 19

The peptide bond has two resonance forms that contribute some double-bond character and inhibit rotation around its axis, so that the alpha carbons are roughly coplanar. The other two dihedral angles in the peptide bond determine the local shape assumed by the protein backbone. [25] : 31 The end with a free amino group is known as the N-terminus or amino terminus, whereas the end of the protein with a free carboxyl group is known as the C-terminus or carboxy terminus (the sequence of the protein is written from N-terminus to C-terminus, from left to right).

The words protein, polypeptide, and peptide are a little ambiguous and can overlap in meaning. Protein is generally used to refer to the complete biological molecule in a stable conformation, whereas peptide is generally reserved for a short amino acid oligomers often lacking a stable 3D structure. But the boundary between the two is not well defined and usually lies near 20–30 residues. [26] Polypeptide can refer to any single linear chain of amino acids, usually regardless of length, but often implies an absence of a defined conformation.


Abundance in cells

It has been estimated that average-sized bacteria contain about 2 million proteins per cell (e.g. E. coli and Staphylococcus aureus). Smaller bacteria, such as Mycoplasma or spirochetes contain fewer molecules, on the order of 50,000 to 1 million. By contrast, eukaryotic cells are larger and thus contain much more protein. For instance, yeast cells have been estimated to contain about 50 million proteins and human cells on the order of 1 to 3 billion. [30] The concentration of individual protein copies ranges from a few molecules per cell up to 20 million. [31] Not all genes coding proteins are expressed in most cells and their number depends on, for example, cell type and external stimuli. For instance, of the 20,000 or so proteins encoded by the human genome, only 6,000 are detected in lymphoblastoid cells. [32]


Proteins are assembled from amino acids using information encoded in genes. Each protein has its own unique amino acid sequence that is specified by the nucleotide sequence of the gene encoding this protein. The genetic code is a set of three-nucleotide sets called codons and each three-nucleotide combination designates an amino acid, for example AUG (adenine–uracil–guanine) is the code for methionine. Because DNA contains four nucleotides, the total number of possible codons is 64 hence, there is some redundancy in the genetic code, with some amino acids specified by more than one codon. [29] : 1002–42 Genes encoded in DNA are first transcribed into pre-messenger RNA (mRNA) by proteins such as RNA polymerase. Most organisms then process the pre-mRNA (also known as a primary transcript) using various forms of Post-transcriptional modification to form the mature mRNA, which is then used as a template for protein synthesis by the ribosome. In prokaryotes the mRNA may either be used as soon as it is produced, or be bound by a ribosome after having moved away from the nucleoid. In contrast, eukaryotes make mRNA in the cell nucleus and then translocate it across the nuclear membrane into the cytoplasm, where protein synthesis then takes place. The rate of protein synthesis is higher in prokaryotes than eukaryotes and can reach up to 20 amino acids per second. [33]

The process of synthesizing a protein from an mRNA template is known as translation. The mRNA is loaded onto the ribosome and is read three nucleotides at a time by matching each codon to its base pairing anticodon located on a transfer RNA molecule, which carries the amino acid corresponding to the codon it recognizes. The enzyme aminoacyl tRNA synthetase "charges" the tRNA molecules with the correct amino acids. The growing polypeptide is often termed the nascent chain. Proteins are always biosynthesized from N-terminus to C-terminus. [29] : 1002–42

The size of a synthesized protein can be measured by the number of amino acids it contains and by its total molecular mass, which is normally reported in units of daltons (synonymous with atomic mass units), or the derivative unit kilodalton (kDa). The average size of a protein increases from Archaea to Bacteria to Eukaryote (283, 311, 438 residues and 31, 34, 49 kDa respectively) due to a bigger number of protein domains constituting proteins in higher organisms. [34] For instance, yeast proteins are on average 466 amino acids long and 53 kDa in mass. [26] The largest known proteins are the titins, a component of the muscle sarcomere, with a molecular mass of almost 3,000 kDa and a total length of almost 27,000 amino acids. [35]

Chemical synthesis

Short proteins can also be synthesized chemically by a family of methods known as peptide synthesis, which rely on organic synthesis techniques such as chemical ligation to produce peptides in high yield. [36] Chemical synthesis allows for the introduction of non-natural amino acids into polypeptide chains, such as attachment of fluorescent probes to amino acid side chains. [37] These methods are useful in laboratory biochemistry and cell biology, though generally not for commercial applications. Chemical synthesis is inefficient for polypeptides longer than about 300 amino acids, and the synthesized proteins may not readily assume their native tertiary structure. Most chemical synthesis methods proceed from C-terminus to N-terminus, opposite the biological reaction. [38]

Most proteins fold into unique 3D structures. The shape into which a protein naturally folds is known as its native conformation. [25] : 36 Although many proteins can fold unassisted, simply through the chemical properties of their amino acids, others require the aid of molecular chaperones to fold into their native states. [25] : 37 Biochemists often refer to four distinct aspects of a protein's structure: [25] : 30–34

  • Primary structure: the amino acid sequence. A protein is a polyamide.
  • Secondary structure: regularly repeating local structures stabilized by hydrogen bonds. The most common examples are the α-helix, β-sheet and turns. Because secondary structures are local, many regions of different secondary structure can be present in the same protein molecule.
  • Tertiary structure: the overall shape of a single protein molecule the spatial relationship of the secondary structures to one another. Tertiary structure is generally stabilized by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through salt bridges, hydrogen bonds, disulfide bonds, and even posttranslational modifications. The term "tertiary structure" is often used as synonymous with the term fold. The tertiary structure is what controls the basic function of the protein.
  • Quaternary structure: the structure formed by several protein molecules (polypeptide chains), usually called protein subunits in this context, which function as a single protein complex.
  • Quinary structure: the signatures of protein surface that organize the crowded cellular interior. Quinary structure is dependent on transient, yet essential, macromolecular interactions that occur inside living cells.

Proteins are not entirely rigid molecules. In addition to these levels of structure, proteins may shift between several related structures while they perform their functions. In the context of these functional rearrangements, these tertiary or quaternary structures are usually referred to as "conformations", and transitions between them are called conformational changes. Such changes are often induced by the binding of a substrate molecule to an enzyme's active site, or the physical region of the protein that participates in chemical catalysis. In solution proteins also undergo variation in structure through thermal vibration and the collision with other molecules. [29] : 368–75

Proteins can be informally divided into three main classes, which correlate with typical tertiary structures: globular proteins, fibrous proteins, and membrane proteins. Almost all globular proteins are soluble and many are enzymes. Fibrous proteins are often structural, such as collagen, the major component of connective tissue, or keratin, the protein component of hair and nails. Membrane proteins often serve as receptors or provide channels for polar or charged molecules to pass through the cell membrane. [29] : 165–85

A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack and hence promoting their own dehydration, are called dehydrons. [39]

Protein domains

Many proteins are composed of several protein domains, i.e. segments of a protein that fold into distinct structural units. Domains usually also have specific functions, such as enzymatic activities (e.g. kinase) or they serve as binding modules (e.g. the SH3 domain binds to proline-rich sequences in other proteins).

Sequence motif

Short amino acid sequences within proteins often act as recognition sites for other proteins. [40] For instance, SH3 domains typically bind to short PxxP motifs (i.e. 2 prolines [P], separated by two unspecified amino acids [x], although the surrounding amino acids may determine the exact binding specificity). Many such motifs has been collected in the Eukaryotic Linear Motif (ELM) database.

Proteins are the chief actors within the cell, said to be carrying out the duties specified by the information encoded in genes. [26] With the exception of certain types of RNA, most other biological molecules are relatively inert elements upon which proteins act. Proteins make up half the dry weight of an Escherichia coli cell, whereas other macromolecules such as DNA and RNA make up only 3% and 20%, respectively. [41] The set of proteins expressed in a particular cell or cell type is known as its proteome.

The chief characteristic of proteins that also allows their diverse set of functions is their ability to bind other molecules specifically and tightly. The region of the protein responsible for binding another molecule is known as the binding site and is often a depression or "pocket" on the molecular surface. This binding ability is mediated by the tertiary structure of the protein, which defines the binding site pocket, and by the chemical properties of the surrounding amino acids' side chains. Protein binding can be extraordinarily tight and specific for example, the ribonuclease inhibitor protein binds to human angiogenin with a sub-femtomolar dissociation constant (<10 −15 M) but does not bind at all to its amphibian homolog onconase (>1 M). Extremely minor chemical changes such as the addition of a single methyl group to a binding partner can sometimes suffice to nearly eliminate binding for example, the aminoacyl tRNA synthetase specific to the amino acid valine discriminates against the very similar side chain of the amino acid isoleucine. [42]

Proteins can bind to other proteins as well as to small-molecule substrates. When proteins bind specifically to other copies of the same molecule, they can oligomerize to form fibrils this process occurs often in structural proteins that consist of globular monomers that self-associate to form rigid fibers. Protein–protein interactions also regulate enzymatic activity, control progression through the cell cycle, and allow the assembly of large protein complexes that carry out many closely related reactions with a common biological function. Proteins can also bind to, or even be integrated into, cell membranes. The ability of binding partners to induce conformational changes in proteins allows the construction of enormously complex signaling networks. [29] : 830–49 As interactions between proteins are reversible, and depend heavily on the availability of different groups of partner proteins to form aggregates that are capable to carry out discrete sets of function, study of the interactions between specific proteins is a key to understand important aspects of cellular function, and ultimately the properties that distinguish particular cell types. [43] [44]


The best-known role of proteins in the cell is as enzymes, which catalyse chemical reactions. Enzymes are usually highly specific and accelerate only one or a few chemical reactions. Enzymes carry out most of the reactions involved in metabolism, as well as manipulating DNA in processes such as DNA replication, DNA repair, and transcription. Some enzymes act on other proteins to add or remove chemical groups in a process known as posttranslational modification. About 4,000 reactions are known to be catalysed by enzymes. [45] The rate acceleration conferred by enzymatic catalysis is often enormous—as much as 10 17 -fold increase in rate over the uncatalysed reaction in the case of orotate decarboxylase (78 million years without the enzyme, 18 milliseconds with the enzyme). [46]

The molecules bound and acted upon by enzymes are called substrates. Although enzymes can consist of hundreds of amino acids, it is usually only a small fraction of the residues that come in contact with the substrate, and an even smaller fraction—three to four residues on average—that are directly involved in catalysis. [47] The region of the enzyme that binds the substrate and contains the catalytic residues is known as the active site.

Dirigent proteins are members of a class of proteins that dictate the stereochemistry of a compound synthesized by other enzymes. [48]

Cell signaling and ligand binding

Many proteins are involved in the process of cell signaling and signal transduction. Some proteins, such as insulin, are extracellular proteins that transmit a signal from the cell in which they were synthesized to other cells in distant tissues. Others are membrane proteins that act as receptors whose main function is to bind a signaling molecule and induce a biochemical response in the cell. Many receptors have a binding site exposed on the cell surface and an effector domain within the cell, which may have enzymatic activity or may undergo a conformational change detected by other proteins within the cell. [28] : 251–81

Antibodies are protein components of an adaptive immune system whose main function is to bind antigens, or foreign substances in the body, and target them for destruction. Antibodies can be secreted into the extracellular environment or anchored in the membranes of specialized B cells known as plasma cells. Whereas enzymes are limited in their binding affinity for their substrates by the necessity of conducting their reaction, antibodies have no such constraints. An antibody's binding affinity to its target is extraordinarily high. [29] : 275–50

Many ligand transport proteins bind particular small biomolecules and transport them to other locations in the body of a multicellular organism. These proteins must have a high binding affinity when their ligand is present in high concentrations, but must also release the ligand when it is present at low concentrations in the target tissues. The canonical example of a ligand-binding protein is haemoglobin, which transports oxygen from the lungs to other organs and tissues in all vertebrates and has close homologs in every biological kingdom. [29] : 222–29 Lectins are sugar-binding proteins which are highly specific for their sugar moieties. Lectins typically play a role in biological recognition phenomena involving cells and proteins. [49] Receptors and hormones are highly specific binding proteins.

Transmembrane proteins can also serve as ligand transport proteins that alter the permeability of the cell membrane to small molecules and ions. The membrane alone has a hydrophobic core through which polar or charged molecules cannot diffuse. Membrane proteins contain internal channels that allow such molecules to enter and exit the cell. Many ion channel proteins are specialized to select for only a particular ion for example, potassium and sodium channels often discriminate for only one of the two ions. [28] : 232–34

Structural proteins

Structural proteins confer stiffness and rigidity to otherwise-fluid biological components. Most structural proteins are fibrous proteins for example, collagen and elastin are critical components of connective tissue such as cartilage, and keratin is found in hard or filamentous structures such as hair, nails, feathers, hooves, and some animal shells. [29] : 178–81 Some globular proteins can also play structural functions, for example, actin and tubulin are globular and soluble as monomers, but polymerize to form long, stiff fibers that make up the cytoskeleton, which allows the cell to maintain its shape and size.

Other proteins that serve structural functions are motor proteins such as myosin, kinesin, and dynein, which are capable of generating mechanical forces. These proteins are crucial for cellular motility of single celled organisms and the sperm of many multicellular organisms which reproduce sexually. They also generate the forces exerted by contracting muscles [29] : 258–64, 272 and play essential roles in intracellular transport.

A key question in molecular biology is how proteins evolve, i.e. how can mutations (or rather changes in amino acid sequence) lead to new structures and functions? Most amino acids in a protein can be changed without disrupting activity or function, as can be seen from numerous homologous proteins across species (as collected in specialized databases for protein families, e.g. PFAM). [50] In order to prevent dramatic consequences of mutations, a gene may be duplicated before it can mutate freely. However, this can also lead to complete loss of gene function and thus pseudo-genes. [51] More commonly, single amino acid changes have limited consequences although some can change protein function substantially, especially in enzymes. For instance, many enzymes can change their substrate specificity by one or a few mutations. [52] Changes in substrate specificity are facilitated by substrate promiscuity, i.e. the ability of many enzymes to bind and process multiple substrates. When mutations occur, the specificity of an enzyme can increase (or decrease) and thus its enzymatic activity. [52] Thus, bacteria (or other organisms) can adapt to different food sources, including unnatural substrates such as plastic. [53]

The activities and structures of proteins may be examined in vitro, in vivo, and in silico. In vitro studies of purified proteins in controlled environments are useful for learning how a protein carries out its function: for example, enzyme kinetics studies explore the chemical mechanism of an enzyme's catalytic activity and its relative affinity for various possible substrate molecules. By contrast, in vivo experiments can provide information about the physiological role of a protein in the context of a cell or even a whole organism. In silico studies use computational methods to study proteins.

Protein purification

To perform in vitro analysis, a protein must be purified away from other cellular components. This process usually begins with cell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins membrane lipids and proteins cellular organelles, and nucleic acids. Precipitation by a method known as salting out can concentrate the proteins from this lysate. Various types of chromatography are then used to isolate the protein or proteins of interest based on properties such as molecular weight, net charge and binding affinity. [25] : 21–24 The level of purification can be monitored using various types of gel electrophoresis if the desired protein's molecular weight and isoelectric point are known, by spectroscopy if the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity. Additionally, proteins can be isolated according to their charge using electrofocusing. [54]

For natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process, genetic engineering is often used to add chemical features to proteins that make them easier to purify without affecting their structure or activity. Here, a "tag" consisting of a specific amino acid sequence, often a series of histidine residues (a "His-tag"), is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containing nickel, the histidine residues ligate the nickel and attach to the column while the untagged components of the lysate pass unimpeded. A number of different tags have been developed to help researchers purify specific proteins from complex mixtures. [55]

Cellular localization

The study of proteins in vivo is often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in the cytoplasm and membrane-bound or secreted proteins in the endoplasmic reticulum, the specifics of how proteins are targeted to specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell a fusion protein or chimera consisting of the natural protein of interest linked to a "reporter" such as green fluorescent protein (GFP). [56] The fused protein's position within the cell can be cleanly and efficiently visualized using microscopy, [57] as shown in the figure opposite.

Other methods for elucidating the cellular location of proteins requires the use of known compartmental markers for regions such as the ER, the Golgi, lysosomes or vacuoles, mitochondria, chloroplasts, plasma membrane, etc. With the use of fluorescently tagged versions of these markers or of antibodies to known markers, it becomes much simpler to identify the localization of a protein of interest. For example, indirect immunofluorescence will allow for fluorescence colocalization and demonstration of location. Fluorescent dyes are used to label cellular compartments for a similar purpose. [58]

Other possibilities exist, as well. For example, immunohistochemistry usually utilizes an antibody to one or more proteins of interest that are conjugated to enzymes yielding either luminescent or chromogenic signals that can be compared between samples, allowing for localization information. Another applicable technique is cofractionation in sucrose (or other material) gradients using isopycnic centrifugation. [59] While this technique does not prove colocalization of a compartment of known density and the protein of interest, it does increase the likelihood, and is more amenable to large-scale studies.

Finally, the gold-standard method of cellular localization is immunoelectron microscopy. This technique also uses an antibody to the protein of interest, along with classical electron microscopy techniques. The sample is prepared for normal electron microscopic examination, and then treated with an antibody to the protein of interest that is conjugated to an extremely electro-dense material, usually gold. This allows for the localization of both ultrastructural details as well as the protein of interest. [60]

Through another genetic engineering application known as site-directed mutagenesis, researchers can alter the protein sequence and hence its structure, cellular localization, and susceptibility to regulation. This technique even allows the incorporation of unnatural amino acids into proteins, using modified tRNAs, [61] and may allow the rational design of new proteins with novel properties. [62]


The total complement of proteins present at a time in a cell or cell type is known as its proteome, and the study of such large-scale data sets defines the field of proteomics, named by analogy to the related field of genomics. Key experimental techniques in proteomics include 2D electrophoresis, [63] which allows the separation of many proteins, mass spectrometry, [64] which allows rapid high-throughput identification of proteins and sequencing of peptides (most often after in-gel digestion), protein microarrays, which allow the detection of the relative levels of the various proteins present in a cell, and two-hybrid screening, which allows the systematic exploration of protein–protein interactions. [65] The total complement of biologically possible such interactions is known as the interactome. [66] A systematic attempt to determine the structures of proteins representing every possible fold is known as structural genomics. [67]

Structure determination

Discovering the tertiary structure of a protein, or the quaternary structure of its complexes, can provide important clues about how the protein performs its function and how it can be affected, i.e. in drug design. As proteins are too small to be seen under a light microscope, other methods have to be employed to determine their structure. Common experimental methods include X-ray crystallography and NMR spectroscopy, both of which can produce structural information at atomic resolution. However, NMR experiments are able to provide information from which a subset of distances between pairs of atoms can be estimated, and the final possible conformations for a protein are determined by solving a distance geometry problem. Dual polarisation interferometry is a quantitative analytical method for measuring the overall protein conformation and conformational changes due to interactions or other stimulus. Circular dichroism is another laboratory technique for determining internal β-sheet / α-helical composition of proteins. Cryoelectron microscopy is used to produce lower-resolution structural information about very large protein complexes, including assembled viruses [28] : 340–41 a variant known as electron crystallography can also produce high-resolution information in some cases, especially for two-dimensional crystals of membrane proteins. [68] Solved structures are usually deposited in the Protein Data Bank (PDB), a freely available resource from which structural data about thousands of proteins can be obtained in the form of Cartesian coordinates for each atom in the protein. [69]

Many more gene sequences are known than protein structures. Further, the set of solved structures is biased toward proteins that can be easily subjected to the conditions required in X-ray crystallography, one of the major structure determination methods. In particular, globular proteins are comparatively easy to crystallize in preparation for X-ray crystallography. Membrane proteins and large protein complexes, by contrast, are difficult to crystallize and are underrepresented in the PDB. [70] Structural genomics initiatives have attempted to remedy these deficiencies by systematically solving representative structures of major fold classes. Protein structure prediction methods attempt to provide a means of generating a plausible structure for proteins whose structures have not been experimentally determined. [71]

Structure prediction

Complementary to the field of structural genomics, protein structure prediction develops efficient mathematical models of proteins to computationally predict the molecular formations in theory, instead of detecting structures with laboratory observation. [72] The most successful type of structure prediction, known as homology modeling, relies on the existence of a "template" structure with sequence similarity to the protein being modeled structural genomics' goal is to provide sufficient representation in solved structures to model most of those that remain. [73] Although producing accurate models remains a challenge when only distantly related template structures are available, it has been suggested that sequence alignment is the bottleneck in this process, as quite accurate models can be produced if a "perfect" sequence alignment is known. [74] Many structure prediction methods have served to inform the emerging field of protein engineering, in which novel protein folds have already been designed. [75] Also proteins (in eukaryotes

33%) contain large unstructured but biologically functional segments and can be classified as intrinsically disordered proteins. [76] Predicting and analysing protein disorder is, therefore, an important part of protein structure characterisation. [77]


A vast array of computational methods have been developed to analyze the structure, function and evolution of proteins. The development of such tools has been driven by the large amount of genomic and proteomic data available for a variety of organisms, including the human genome. It is simply impossible to study all proteins experimentally, hence only a few are subjected to laboratory experiments while computational tools are used to extrapolate to similar proteins. Such homologous proteins can be efficiently identified in distantly related organisms by sequence alignment. Genome and gene sequences can be searched by a variety of tools for certain properties. Sequence profiling tools can find restriction enzyme sites, open reading frames in nucleotide sequences, and predict secondary structures. Phylogenetic trees can be constructed and evolutionary hypotheses developed using special software like ClustalW regarding the ancestry of modern organisms and the genes they express. The field of bioinformatics is now indispensable for the analysis of genes and proteins.

In silico simulation of dynamical processes

A more complex computational problem is the prediction of intermolecular interactions, such as in molecular docking, [78] protein folding, protein–protein interaction and chemical reactivity. Mathematical models to simulate these dynamical processes involve molecular mechanics, in particular, molecular dynamics. In this regard, in silico simulations discovered the folding of small α-helical protein domains such as the villin headpiece, [79] the HIV accessory protein [80] and hybrid methods combining standard molecular dynamics with quantum mechanical mathematics have explored the electronic states of rhodopsins. [81]

Beyond classical molecular dynamics, quantum dynamics methods allow the simulation of proteins in atomistic detail with an accurate description of quantum mechanical effects. Examples include the multi-layer multi-configuration time-dependent Hartree (MCTDH) method and the hierarchical equations of motion (HEOM) approach, which have been applied to plant cryptochromes [82] and bacteria light-harvesting complexes, [83] respectively. Both quantum and classical mechanical simulations of biological-scale systems are extremely computationally demanding, so distributed computing initiatives (for example, the [email protected] project [84] ) facilitate the molecular modeling by exploiting advances in GPU parallel processing and Monte Carlo techniques.

Chemical analysis

The total nitrogen content of organic matter is mainly formed by the amino groups in proteins. The Total Kjeldahl Nitrogen (TKN) is a measure of nitrogen widely used in the analysis of (waste) water, soil, food, feed and organic matter in general. As the name suggests, the Kjeldahl method is applied. More sensitive methods are available. [85] [86]

Most microorganisms and plants can biosynthesize all 20 standard amino acids, while animals (including humans) must obtain some of the amino acids from the diet. [41] The amino acids that an organism cannot synthesize on its own are referred to as essential amino acids. Key enzymes that synthesize certain amino acids are not present in animals—such as aspartokinase, which catalyses the first step in the synthesis of lysine, methionine, and threonine from aspartate. If amino acids are present in the environment, microorganisms can conserve energy by taking up the amino acids from their surroundings and downregulating their biosynthetic pathways.

In animals, amino acids are obtained through the consumption of foods containing protein. Ingested proteins are then broken down into amino acids through digestion, which typically involves denaturation of the protein through exposure to acid and hydrolysis by enzymes called proteases. Some ingested amino acids are used for protein biosynthesis, while others are converted to glucose through gluconeogenesis, or fed into the citric acid cycle. This use of protein as a fuel is particularly important under starvation conditions as it allows the body's own proteins to be used to support life, particularly those found in muscle. [87]

In animals such as dogs and cats, protein maintains the health and quality of the skin by promoting hair follicle growth and keratinization, and thus reducing the likelihood of skin problems producing malodours. [88] Poor-quality proteins also have a role regarding gastrointestinal health, increasing the potential for flatulence and odorous compounds in dogs because when proteins reach the colon in an undigested state, they are fermented producing hydrogen sulfide gas, indole, and skatole. [89] Dogs and cats digest animal proteins better than those from plants, but products of low-quality animal origin are poorly digested, including skin, feathers, and connective tissue. [89]


The authors thank Dima Kozakov for providing access to the ClusPro server and Olivier Michielin and VIncent Zoete for providing access to the SwissDock server.

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In the early 1930s, William Astbury showed that there were drastic changes in the X-ray fiber diffraction of moist wool or hair fibers upon significant stretching. The data suggested that the unstretched fibers had a coiled molecular structure with a characteristic repeat of ≈5.1 ångströms (0.51 nanometres).

Astbury initially proposed a linked-chain structure for the fibers. He later joined other researchers (notably the American chemist Maurice Huggins) in proposing that:

  • the unstretched protein molecules formed a helix (which he called the α-form)
  • the stretching caused the helix to uncoil, forming an extended state (which he called the β-form).

Although incorrect in their details, Astbury's models of these forms were correct in essence and correspond to modern elements of secondary structure, the α-helix and the β-strand (Astbury's nomenclature was kept), which were developed by Linus Pauling, Robert Corey and Herman Branson in 1951 (see below) that paper showed both right- and left-handed helices, although in 1960 the crystal structure of myoglobin [1] showed that the right-handed form is the common one. Hans Neurath was the first to show that Astbury's models could not be correct in detail, because they involved clashes of atoms. [2] Neurath's paper and Astbury's data inspired H. S. Taylor, [3] Maurice Huggins [4] and Bragg and collaborators [5] to propose models of keratin that somewhat resemble the modern α-helix.

Two key developments in the modeling of the modern α-helix were: the correct bond geometry, thanks to the crystal structure determinations of amino acids and peptides and Pauling's prediction of planar peptide bonds and his relinquishing of the assumption of an integral number of residues per turn of the helix. The pivotal moment came in the early spring of 1948, when Pauling caught a cold and went to bed. Being bored, he drew a polypeptide chain of roughly correct dimensions on a strip of paper and folded it into a helix, being careful to maintain the planar peptide bonds. After a few attempts, he produced a model with physically plausible hydrogen bonds. Pauling then worked with Corey and Branson to confirm his model before publication. [6] In 1954, Pauling was awarded his first Nobel Prize "for his research into the nature of the chemical bond and its application to the elucidation of the structure of complex substances" [7] (such as proteins), prominently including the structure of the α-helix.

Geometry and hydrogen bonding Edit

The amino acids in an α-helix are arranged in a right-handed helical structure where each amino acid residue corresponds to a 100° turn in the helix (i.e., the helix has 3.6 residues per turn), and a translation of 1.5 Å (0.15 nm) along the helical axis. Dunitz [8] describes how Pauling's first article on the theme in fact shows a left-handed helix, the enantiomer of the true structure. Short pieces of left-handed helix sometimes occur with a large content of achiral glycine amino acids, but are unfavorable for the other normal, biological L -amino acids. The pitch of the alpha-helix (the vertical distance between consecutive turns of the helix) is 5.4 Å (0.54 nm), which is the product of 1.5 and 3.6. What is most important is that the N-H group of an amino acid forms a hydrogen bond with the C=O group of the amino acid four residues earlier this repeated i + 4 → i hydrogen bonding is the most prominent characteristic of an α-helix. Official international nomenclature [9] [10] specifies two ways of defining α-helices, rule 6.2 in terms of repeating φ, ψ torsion angles (see below) and rule 6.3 in terms of the combined pattern of pitch and hydrogen bonding. The α-helices can be identified in protein structure using several computational methods, one of which is DSSP (Define Secondary Structure of Protein). [11]

Similar structures include the 310 helix (i + 3 → i hydrogen bonding) and the π-helix (i + 5 → i hydrogen bonding). The α-helix can be described as a 3.613 helix, since the i + 4 spacing adds three more atoms to the H-bonded loop compared to the tighter 310 helix, and on average, 3.6 amino acids are involved in one ring of α-helix. The subscripts refer to the number of atoms (including the hydrogen) in the closed loop formed by the hydrogen bond. [12]

Residues in α-helices typically adopt backbone (φ, ψ) dihedral angles around (−60°, −45°), as shown in the image at right. In more general terms, they adopt dihedral angles such that the ψ dihedral angle of one residue and the φ dihedral angle of the next residue sum to roughly −105°. As a consequence, α-helical dihedral angles, in general, fall on a diagonal stripe on the Ramachandran diagram (of slope −1), ranging from (−90°, −15°) to (−35°, −70°). For comparison, the sum of the dihedral angles for a 310 helix is roughly −75°, whereas that for the π-helix is roughly −130°. The general formula for the rotation angle Ω per residue of any polypeptide helix with trans isomers is given by the equation [14] [15]

3 cos Ω = 1 − 4 cos 2 φ + ψ / 2

The α-helix is tightly packed there is almost no free space within the helix. The amino-acid side-chains are on the outside of the helix, and point roughly "downward" (i.e., toward the N-terminus), like the branches of an evergreen tree (Christmas tree effect). This directionality is sometimes used in preliminary, low-resolution electron-density maps to determine the direction of the protein backbone. [16]

Stability Edit

Helices observed in proteins can range from four to over forty residues long, but a typical helix contains about ten amino acids (about three turns). In general, short polypeptides do not exhibit much α-helical structure in solution, since the entropic cost associated with the folding of the polypeptide chain is not compensated for by a sufficient amount of stabilizing interactions. In general, the backbone hydrogen bonds of α-helices are considered slightly weaker than those found in β-sheets, and are readily attacked by the ambient water molecules. However, in more hydrophobic environments such as the plasma membrane, or in the presence of co-solvents such as trifluoroethanol (TFE), or isolated from solvent in the gas phase, [17] oligopeptides readily adopt stable α-helical structure. Furthermore, crosslinks can be incorporated into peptides to conformationally stabilize helical folds. Crosslinks stabilize the helical state by entropically destabilizing the unfolded state and by removing enthalpically stabilized "decoy" folds that compete with the fully helical state. [18] It has been shown that α-helices are more stable, robust to mutations and designable than β-strands in natural proteins, [19] and also in artificial designed proteins. [20]

Since the α-helix is defined by its hydrogen bonds and backbone conformation, the most detailed experimental evidence for α-helical structure comes from atomic-resolution X-ray crystallography such as the example shown at right. It is clear that all the backbone carbonyl oxygens point downward (toward the C-terminus) but splay out slightly, and the H-bonds are approximately parallel to the helix axis. Protein structures from NMR spectroscopy also show helices well, with characteristic observations of nuclear Overhauser effect (NOE) couplings between atoms on adjacent helical turns. In some cases, the individual hydrogen bonds can be observed directly as a small scalar coupling in NMR.

There are several lower-resolution methods for assigning general helical structure. The NMR chemical shifts (in particular of the C α , C β and C′) and residual dipolar couplings are often characteristic of helices. The far-UV (170–250 nm) circular dichroism spectrum of helices is also idiosyncratic, exhibiting a pronounced double minimum at around 208 and 222 nm. Infrared spectroscopy is rarely used, since the α-helical spectrum resembles that of a random coil (although these might be discerned by, e.g., hydrogen-deuterium exchange). Finally, cryo electron microscopy is now capable of discerning individual α-helices within a protein, although their assignment to residues is still an active area of research.

Long homopolymers of amino acids often form helices if soluble. Such long, isolated helices can also be detected by other methods, such as dielectric relaxation, flow birefringence, and measurements of the diffusion constant. In stricter terms, these methods detect only the characteristic prolate (long cigar-like) hydrodynamic shape of a helix, or its large dipole moment.

Different amino-acid sequences have different propensities for forming α-helical structure. Methionine, alanine, leucine, glutamate, and lysine uncharged ("MALEK" in the amino-acid 1-letter codes) all have especially high helix-forming propensities, whereas proline and glycine have poor helix-forming propensities. [21] Proline either breaks or kinks a helix, both because it cannot donate an amide hydrogen bond (having no amide hydrogen), and also because its sidechain interferes sterically with the backbone of the preceding turn – inside a helix, this forces a bend of about 30° in the helix's axis. [12] However, proline is often seen as the first residue of a helix, it is presumed due to its structural rigidity. At the other extreme, glycine also tends to disrupt helices because its high conformational flexibility makes it entropically expensive to adopt the relatively constrained α-helical structure.

Table of standard amino acid alpha-helical propensities Edit

Estimated differences in free energy, Δ(ΔG), estimated in kcal/mol per residue in an α-helical configuration, relative to alanine arbitrarily set as zero. Higher numbers (more positive free energies) are less favoured. Significant deviations from these average numbers are possible, depending on the identities of the neighbouring residues.

Differences in free energy per residue [22]
Amino acid 3-
Helical penalty
kcal/mol kJ/mol
Alanine Ala A 0.00 0.00
Arginine Arg R 0.21 0.88
Asparagine Asn N 0.65 2.72
Aspartic acid Asp D 0.69 2.89
Cysteine Cys C 0.68 2.85
Glutamic acid Glu E 0.40 1.67
Glutamine Gln Q 0.39 1.63
Glycine Gly G 1.00 4.18
Histidine His H 0.61 2.55
Isoleucine Ile I 0.41 1.72
Leucine Leu L 0.21 0.88
Lysine Lys K 0.26 1.09
Methionine Met M 0.24 1.00
Phenylalanine Phe F 0.54 2.26
Proline Pro P 3.16 13.22
Serine Ser S 0.50 2.09
Threonine Thr T 0.66 2.76
Tryptophan Trp W 0.49 2.05
Tyrosine Tyr Y 0.53 2.22
Valine Val V 0.61 2.55

A helix has an overall dipole moment due to the aggregate effect of the individual microdipoles from the carbonyl groups of the peptide bond pointing along the helix axis. [23] The effects of this macrodipole are a matter of some controversy. α-helices often occur with the N-terminal end bound by a negatively charged group, sometimes an amino acid side chain such as glutamate or aspartate, or sometimes a phosphate ion. Some regard the helix macrodipole as interacting electrostatically with such groups. Others feel that this is misleading and it is more realistic to say that the hydrogen bond potential of the free NH groups at the N-terminus of an α-helix can be satisfied by hydrogen bonding this can also be regarded as set of interactions between local microdipoles such as C=O···H−N . [24] [25]

Coiled-coil α helices are highly stable forms in which two or more helices wrap around each other in a "supercoil" structure. Coiled coils contain a highly characteristic sequence motif known as a heptad repeat, in which the motif repeats itself every seven residues along the sequence (amino acid residues, not DNA base-pairs). The first and especially the fourth residues (known as the a and d positions) are almost always hydrophobic the fourth residue is typically leucine – this gives rise to the name of the structural motif called a leucine zipper, which is a type of coiled-coil. These hydrophobic residues pack together in the interior of the helix bundle. In general, the fifth and seventh residues (the e and g positions) have opposing charges and form a salt bridge stabilized by electrostatic interactions. Fibrous proteins such as keratin or the "stalks" of myosin or kinesin often adopt coiled-coil structures, as do several dimerizing proteins. A pair of coiled-coils – a four-helix bundle – is a very common structural motif in proteins. For example, it occurs in human growth hormone and several varieties of cytochrome. The Rop protein, which promotes plasmid replication in bacteria, is an interesting case in which a single polypeptide forms a coiled-coil and two monomers assemble to form a four-helix bundle.

The amino acids that make up a particular helix can be plotted on a helical wheel, a representation that illustrates the orientations of the constituent amino acids (see the article for leucine zipper for such a diagram). Often in globular proteins, as well as in specialized structures such as coiled-coils and leucine zippers, an α-helix will exhibit two "faces" – one containing predominantly hydrophobic amino acids oriented toward the interior of the protein, in the hydrophobic core, and one containing predominantly polar amino acids oriented toward the solvent-exposed surface of the protein.

Changes in binding orientation also occur for facially-organized oligopeptides. This pattern is especially common in antimicrobial peptides, and many models have been devised to describe how this relates to their function. Common to many of them is that the hydrophobic face of the antimicrobial peptide forms pores in the plasma membrane after associating with the fatty chains at the membrane core. [26] [27]

Myoglobin and hemoglobin, the first two proteins whose structures were solved by X-ray crystallography, have very similar folds made up of about 70% α-helix, with the rest being non-repetitive regions, or "loops" that connect the helices. In classifying proteins by their dominant fold, the Structural Classification of Proteins database maintains a large category specifically for all-α proteins.

Hemoglobin then has an even larger-scale quaternary structure, in which the functional oxygen-binding molecule is made up of four subunits.

DNA binding Edit

α-Helices have particular significance in DNA binding motifs, including helix-turn-helix motifs, leucine zipper motifs and zinc finger motifs. This is because of the convenient structural fact that the diameter of an α-helix is about 12 Å (1.2 nm) including an average set of sidechains, about the same as the width of the major groove in B-form DNA, and also because coiled-coil (or leucine zipper) dimers of helices can readily position a pair of interaction surfaces to contact the sort of symmetrical repeat common in double-helical DNA. [28] An example of both aspects is the transcription factor Max (see image at left), which uses a helical coiled coil to dimerize, positioning another pair of helices for interaction in two successive turns of the DNA major groove.

Membrane spanning Edit

α-Helices are also the most common protein structure element that crosses biological membranes (transmembrane protein), [29] it is presumed because the helical structure can satisfy all backbone hydrogen-bonds internally, leaving no polar groups exposed to the membrane if the sidechains are hydrophobic. Proteins are sometimes anchored by a single membrane-spanning helix, sometimes by a pair, and sometimes by a helix bundle, most classically consisting of seven helices arranged up-and-down in a ring such as for rhodopsins (see image at right) or for G protein–coupled receptors (GPCRs). The structural stability between pairs of α-Helical transmembrane domains rely on conserved membrane interhelical packing motifs, for example, the Glycine-xxx-Glycine (or small-xxx-small) motif. [30]

Mechanical properties Edit

α-Helices under axial tensile deformation, a characteristic loading condition that appears in many alpha-helix-rich filaments and tissues, results in a characteristic three-phase behavior of stiff-soft-stiff tangent modulus. [31] Phase I corresponds to the small-deformation regime during which the helix is stretched homogeneously, followed by phase II, in which alpha-helical turns break mediated by the rupture of groups of H-bonds. Phase III is typically associated with large-deformation covalent bond stretching.

Alpha-helices in proteins may have low-frequency accordion-like motion as observed by the Raman spectroscopy [32] and analyzed via the quasi-continuum model. [33] [34] Helices not stabilized by tertiary interactions show dynamic behavior, which can be mainly attributed to helix fraying from the ends. [35]

Homopolymers of amino acids (such as polylysine) can adopt α-helical structure at low temperature that is "melted out" at high temperatures. This helix–coil transition was once thought to be analogous to protein denaturation. The statistical mechanics of this transition can be modeled using an elegant transfer matrix method, characterized by two parameters: the propensity to initiate a helix and the propensity to extend a helix.

At least five artists have made explicit reference to the α-helix in their work: Julie Newdoll in painting and Julian Voss-Andreae, Bathsheba Grossman, Byron Rubin, and Mike Tyka in sculpture.

San Francisco area artist Julie Newdoll, [36] who holds a degree in Microbiology with a minor in art, has specialized in paintings inspired by microscopic images and molecules since 1990. Her painting "Rise of the Alpha Helix" (2003) features human figures arranged in an α helical arrangement. According to the artist, "the flowers reflect the various types of sidechains that each amino acid holds out to the world". [36] This same metaphor is also echoed from the scientist's side: "β sheets do not show a stiff repetitious regularity but flow in graceful, twisting curves, and even the α-helix is regular more in the manner of a flower stem, whose branching nodes show the influence of environment, developmental history, and the evolution of each part to match its own idiosyncratic function." [12]

Julian Voss-Andreae is a German-born sculptor with degrees in experimental physics and sculpture. Since 2001 Voss-Andreae creates "protein sculptures" [37] based on protein structure with the α-helix being one of his preferred objects. Voss-Andreae has made α-helix sculptures from diverse materials including bamboo and whole trees. A monument Voss-Andreae created in 2004 to celebrate the memory of Linus Pauling, the discoverer of the α-helix, is fashioned from a large steel beam rearranged in the structure of the α-helix. The 10-foot-tall (3 m), bright-red sculpture stands in front of Pauling's childhood home in Portland, Oregon.

Ribbon diagrams of α-helices are a prominent element in the laser-etched crystal sculptures of protein structures created by artist Bathsheba Grossman, such as those of insulin, hemoglobin, and DNA polymerase. [38] Byron Rubin is a former protein crystallographer now professional sculptor in metal of proteins, nucleic acids, and drug molecules – many of which featuring α-helices, such as subtilisin, human growth hormone, and phospholipase A2. [39]

Mike Tyka is a computational biochemist at the University of Washington working with David Baker. Tyka has been making sculptures of protein molecules since 2010 from copper and steel, including ubiquitin and a potassium channel tetramer. [40]


The present investigation has explored the influence of the positively charged M2 4′lysine residue upon the function of the recombinant 5-HT3A receptor. The data indicate this residue to be a determinant of the desensitization kinetics, but not the low conductance, of the receptor. Several properties of the receptor were not greatly affected when the 4′K residue was replaced by a series of amino acids that differ in side chain charge and length including arginine (R), glutamine (Q), serine (S) or glycine (G). In those instances, the expressed receptors were functional and displayed rates of activation and Hill coefficients similar to those of the WT 5-HT3A receptor, suggesting that the overall structure of the mutant proteins was not grossly altered.

4′ mutations and single-channel conductance

The single-channel conductance of the 5-HT3 receptor native to neurones and neuronal cell lines varies by approximately 60-fold between preparations (i.e. 0.3-19 pS Lambert et al. 1989 Yang, 1990 Peters et al. 1993 Hussy et al. 1994 Jones & Surprenant, 1994 Zhong et al. 1999) and there is evidence to suggest the expression of receptors with distinct conductances within the same cell (Derkach et al. 1989 Yang et al. 1992 Hussy et al. 1994). Recent studies (Davies et al. 1999) describing a very large difference in the conductance of 5-HT3 receptors assembled from human 5-HT3A (< 1 pS Brown et al. 1998), or human 5-HT3A and 5-HT3B subunits (� pS Davies et al. 1999), provide a potential explanation for such variation.

However, one unresolved issue is why the single-channel conductance of the homo-oligomeric 5-HT3A receptor is so small. The present work eliminates one possibility: the 4′lysine residue that is unique, within the Cys-loop receptor family, to 5-HT3 receptor subunit M2 sequences. The presence of positively charged residues within the pore region of cation-selective ion channels, as exemplified by the Q/R site of AMPA receptors assembled from GluR2 subunits, is known to markedly suppress single-channel conductance (Swanson et al. 1997). By analogy, if the 4′lysine were to reside upon the face of the M2 α-helix lining the channel lumen, the resulting ring of five positive charges would be expected to influence cation flux dramatically. The lack of effect of any of the four amino acid substitutions upon single-channel conductance argues strongly against this possibility. None of the mutant receptors had a single-channel conductance that was significantly different from that of 390 fS found for the WT 5-HT3A receptor ( Fig. 6 , Table 3 ), a value that compares well with previous estimates of 360 fS (Hussy et al. 1994) and 420 fS (Gill et al. 1995) obtained by fluctuation analysis. This negative finding is unlikely to reflect an inability to detect subtle changes in conductance by fluctuation analysis, because we and others have previously employed this technique to demonstrate the inwardly rectifying properties of the 5-HT3A receptor channel and the modulation of its conductance by extracellular divalent cations (Hussy et al. 1994 Brown et al. 1998). That the analysis technique is capable of detecting differences in the conductance of 5-HT3 receptor channels is additionally shown by the channel conductance of 3.7 pS obtained for the receptor endogenous to rat superior cervical ganglion (SCG) neurones ( Table 3 M. J. Gunthorpe & J. A. Peters, unpublished observations). This value is similar to previous estimates of 2.6 pS and 3.4 pS obtained for rat (Yang et al. 1992) and 3.4 pS for mouse (Hussy et al. 1994) SCG neurones, respectively, using fluctuation analysis. Thus it appears that the presence of a charged residue at the 4′ position of the M2 domain does not contribute to the low single-channel conductance of the 5-HT3A receptor. Likewise, the exchange of uncharged alanine and polar serine residues located at the 4′ position of muscle-type nicotinic acetylcholine γ and ε subunits does not contribute to the difference in conductance observed for fetal (㬑2㬡γδ) and adult (㬑2㬡εδ) receptors (Herlitze et al. 1996).

The present observations add support to a spatial arrangement inferred from application of the substituted cysteine accessibility method to nicotinic and GABAA receptor subunits, whereby the 4′ residue faces into the protein interior (Akabas et al. 1994 Xu & Akabas, 1996). It is conceivable, as originally suggested by Maricq et al. (1991), that the 4′lysine forms a salt bridge with an aspartate residue (D265) located within the adjacent M1 sequence. The latter is also unique to the 5-HT3A and 5-HT3B subunit sequences, since other Cys-loop receptors express either serine, alanine or an aliphatic residue at this position. The lack of effect of any of the mutations at the 4′ position upon channel conductance might provide indirect evidence that salt bridge formation does in fact occur. Modelling of the 5-HT3A receptor ion channel suggests that even if the 4′lysine residue is located upon the face of the α-helix opposite to the lumen, it might still have a through space electrostatic effect sufficient to influence channel conductance (M. S. P. Sansom, personal communication). The absence of such an effect implies neutralization of the positive charge through salt bridge formation or, far more less likely in view of the side chain pKa of lysine (negative log of the acid dissociation constant, 10.4 in solution), deprotonation in the hydrophobic interior of the receptor protein.

If the above scenario is correct, the residues present at the 3′ (i.e. F) and 5′ (i.e. I) positions might reasonably be predicted to interact hydrophobically with the adjacent α-helix at either side, allowing the polar residues 2′threonine (2′T) and 6′serine (6′S) to line the pore. Thus, it is perhaps unsurprising that changing 3𠌯 to either glutamate (E), or lysine, and 5′I to glutamate, resulted in either complete lack or very low levels of binding of the selective antagonist [ 3 H]granisetron to membranes prepared from transfected cells. Interestingly, retention of binding was observed when the 5′I was replaced by lysine. This suggests that the structural changes introduced by the charged residues are better tolerated by the protein at this position, although only lysine, perhaps because it is a more flexible residue than glutamate, can presumably be accommodated. However the resulting receptors appeared to be non-functional, suggesting that either these lysine residues at least partly face the pore in the open channel conformation and prevent ion flow, or that in these mutant receptors the channel cannot actually open.

4′ mutations and agonist sensitivity

Irrespective of the substitution made, a small (approximately 2-fold) decrease in the EC50 for 5-HT was observed. Given the location of the residue, this effect is unlikely to result from direct influence upon the binding site for 5-HT. Instead, M2 mutants of the 5-HT3A and other transmitter-gated channels (e.g. Revah et al. 1991 Yakel et al. 1993) may produce an alteration in the stability of open versus closed states of the receptor (Filatov & White, 1995 Labarca et al. 1995). Such effects on receptor function have, in some instances, been correlated with the physico-chemical character of the amino acid substituted. For example, in a study of recombinant 5-HT3A receptors expressed in Xenopus oocytes, polar substitutions at 9′leucine (9′L) resulted in receptors with enhanced agonist sensitivity (EC50, 0.3-0.6 μ m ) compared to either WT (1.4 μ m ) or hydrophobic substitutions (1𠄱.3 μ m Yakel et al. 1993). A similar rank order of effect was found for equivalent substitutions in the nicotinic 㬗 nACh receptor (Revah et al. 1991). By contrast, all four 4′K substitutions examined in the present study, although involving amino acids with very different properties, produced a similar increase in the potency of 5-HT.

4′K mutations and desensitization

WT and 5-HT3A receptors mutated at the 4′K position all desensitized completely in the prolonged presence of a supramaximal concentration of agonist, but the rate of current decay was substantially slower for all four mutant subunits. It has been reported (Yakel et al. 1993) that the rate of desensitization of WT receptors is accelerated by the presence of divalent cations, particularly [Ca 2+ ], in the extracellular solution (but see Boddeke et al. 1996). In the present study, the macroscopic current response in ‘zero’ calcium solution ([Ca 2+ ]o� nM) desensitized approximately 2-fold more slowly than currents recorded with [Ca 2+ ]o set at 1.8 mM ( Fig. 4 ). Therefore, experiments examining the kinetics of desensitization were conducted in the nominal absence of extracellular Ca 2+ to eliminate a potentially confounding influence that could result from differences in Ca 2+ influx between the mutated channels, although the latter possibility was not addressed specifically.

The rank order of effect of the 4′K substitutions on 5-HT3A receptor desensitization was arginine > glycine > serine > glutamine > lysine (WT) the arginine substitution resulting in more than a 5-fold slower rate of desensitization compared to WT ( Fig. 5 , Table 2 ). Thus, the quantitative effect of the 4′K mutations upon desensitization, unlike agonist potency (see above), is dependent upon the amino acid substituted. However, the effect of the 4′K mutations upon desensitization does not correlate simply with the properties of the side chain of the substituted amino acid. Replacement of the M2 4′lysine residue by the like-charged arginine residue, which has been described as a ‘safe’ substitution in site-directed mutagenesis (Bordo & Argos, 1991), in fact caused the greatest reduction in desensitization rate. In other classes of ion channel, the replacement, by lysine, of arginine residues participating in putative salt bridges that might stabilize protein structure has variable effects. For the cystic fibrosis conductance regulator (CTFR), such substitution within the sixth transmembrane domain (i.e. R347K) is well tolerated and is postulated to maintain the stability of the pore in a high conductance state (Cotten & Walsh, 1999). By contrast, at an inwardly rectifying potassium channel (IRK1), the mutation R148K located within the pore (or H5) region produces non-functional channels (Yang et al. 1997). Yang et al. hypothesize that this mutation disrupts a salt bridge that is exposed in the IRK1 channel. We suggest that such disruption of the putative salt bridge between the 4′ and the M1 D265 residues is less likely within the hydrophobic core of the 5-HT3A receptor protein (see also Cotten & Walsh, 1999). The lack of effect of the mutation upon channel conductance (via a through space electrostatic effect – see above) is consistent with this interpretation. Instead, the slowing of the rate of desensitization produced by the introduction of the arginine residue might be related to the reduced flexibility of this residue compared to lysine, which could result in local structural changes that are necessary to allow the formation of the salt bridge further experiments are necessary to determine if such changes might affect the rate of the conformation change(s) necessary for entry into the desensitized state(s), which might provide an explanation for our data.

If the above interpretation is correct, how can the influence of the remaining amino acid substitutions upon desensitization rate be explained? In the absence of structural information, it might be speculated from the general principles of protein folding that mutations of the 4′ residue that leave the negatively charged D265 residue unpaired in the membrane will lead to a re-orientation within the protein that reduces the energy of this ion within a low dielectric constant (e.g. Perutz, 1979). The conformational freedom available to ‘solvate’ the D265 residue might correlate with the volume of the side chain of the amino acid substituting for the 4′lysine residue. Indeed, the increase in the t1/2 of desensitization does correlate inversely with side chain volume (i.e. Q > S > G). This scheme is, of course, purely conjectural. However, it is interesting to note that changes in the channel gating kinetics of the muscle nACh receptor produced by mutation of a valine residue within the TM3 domain can be related to the volume of the substituted residue (Wang et al. 1999).


In conclusion, the results presented here demonstrate that the lysine residue at the 4′ position in the M2 domain of 5-HT3A receptor has an important role in receptor function but is not the determinant of the low single-channel conductance of this receptor. Most of the biophysical properties of the receptor examined are unaffected by the substitution of lysine with amino acids of differing charge and/or side chain length, but each caused a reduction in the rate of desensitization. Combined with data from the 3𠌯 and 5′I mutants, it would therefore appear that 4′K, and by analogy the 4′ location of other Cys-loop transmitter-gated channels, is not exposed to the channel lumen during the gating or the open state of the channel but it is probably involved in the conformational transition from the open to the desensitized state(s) of the receptor.

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