The primary structure of a segment of a polypeptide chain or of a protein is the amino-acid sequence of the polypeptide chain(s), without regard to spatial arrangement (apart from configuration at the alpha-carbon atom). This definition does not include the positions of disulphide bonds, and is, therefore, not identical with "covalent structure" (IUPAC-IUB, 1970). The commonly occurring amino acids are of 20 different kinds which contain the same dipolar ion group H3N+.CH.COO-. They all have in common a central carbon atom to which are attached a hydrogen atom, an amino group (NH2) and a carboxyl group (COOH). The central carbon atom is called the Calpha-atom and is a chiral centre. All amino acids found in proteins encoded by the genome have the L-configuration at this chiral centre.
This configuration can be remembered as the CORN law. Imagine looking along the H-Calpha bond with the H atom closest to you.
When read clockwise, the groups attached to the Calpha spell the word CORN (Richardson, 1981). There are 20 side chains found in proteins encoded by the genetic machinery of the cell. The side chains confer important properties on a protein such as the ability to bind ligands and catalyse biochemical reactions. They also direct the folding of the nascent polypeptide and stabilise its final conformation. In molecular graphics, atoms can be represented in different ways. For expedience, molecules are often displayed only as lines or vectors between the atoms bonded together covalently. An elegant representation is the ball-and-stick type in which atoms are drawn as coloured spheres and their bonds as rod-like connections. Another useful display is the space-filling representation in which a surface is drawn around the atoms to indicate their van der Waals radii. This surface can be drawn as a series of dots or as a solid entity (Lesk, 1991). Amino acids in proteins(or polypeptides) are joined together by peptide bonds. The sequence of R-groups along the chain is called the primary structure.
The Peptide bond
Pauling et al. (1951) analysed the geometry and dimensions of the peptide bonds in the crystal structures of molecules containing either one or a few peptide bonds. Their results are summarised in the diagram below where the consensus bond lengths are shown in Angstrom units. Bond angles in degrees are also shown for the peptide N and C atoms. It should be noted that the C-N bond length of the peptide is 10% shorter than that found in usual C-N amine bonds (Schulz and Schirmer, 1990; Creighton, 1993).
This is because the peptide bond has some double bond character (40%) due to resonance which occurs with amides. The two canonical structures are:
As a consequence of this resonance all peptide bonds in protein structures are found to be almost planar, i.e. atoms Calpha(i), C(i), O(i), N(i+1) and Calpha(i+1) are approximately co-planar. This rigidity of the peptide bond reduces the degrees of freedom of the polypeptide during folding. The peptide bond nearly always has the trans configuration since it is more favourable than cis, which is sometimes found to occur with proline (Pro) residues (Schulz and Schirmer, 1990).
As can be seen from the previous page, steric hindrance between the functional groups attached to the Calpha atoms will be greater in the cis configuration. However, for proline residues, the cyclic nature of the side chain means that both cis and trans configurations have more equivalent energies. Thus proline is found in the cis configuration more frequently than the other amino acids.
Properties of amino acids
The sequence and properties of side chains determine all that is unique about a particular protein, including its biological function and its specific three-dimensional structure. Histidine (His) is the only side chain that titrates near physiological pH, making it especially useful for enzymatic reactions.
Lysine (Lys) and arginine (Arg) are normally positively charged and aspartate (Asp) and glutamate (Glu) are negatively charged. These charges are very seldom buried in protein interiors except when they are serving some special purpose, as in the activity and activation of chymotrypsin (Blow et al., 1969; Wright, 1973).
Asparagine (Asn) and glutamine (Gln) have interesting hydrogen-bonding properties, since they resemble the backbone peptides. The hydrophobic residues provide a very strong driving force for folding, through the indirect effect of their ceasing to disrupt the water structure once they are buried (Kauzmann, 1959). They also, however, affect the structure in a highly specific manner because their varied sizes and shapes fit together in very efficient packing (Lee and Richards, 1971).
Proline has stronger stereochemical constraints than any other residue, with only one instead of two variable backbone angles, and it lacks the normal backbone NH for hydrogen bonding. It is both disruptive to regular secondary structure and also good at forming turns in the polypeptide chain, so that in spite of its hydrophobicity it is usually found at the edge of the protein (Richardson, 1981).
Glycine (Gly) has three different unique capabilities. As the smallest side group (only a hydrogen), it is often found where main chains approach each other very closely. In addition Gly can assume conformations normally restricted by close contacts of the beta-carbon and finally it is more flexible than other residues, thus contributing to parts of the backbone that need to move or hinge (Richardson, 1981).
Serine (Ser) and threonine (Thr) carry aliphatic hydroxyl groups capable of forming hydrogen bonds with suitable donor or acceptor groups, such as the imino nitrogen or the carbonyl oxygen of the main polypeptide chain. Serine reacts with phosphate by an ester bond, forms part of the catalytic site of many hydrolytic enzymes (Dickerson and Geis, 1969) and contributes to the lining of ion channels. Serine, threonine, and asparagine are also the binding sites of carbohydrates that are attached to the surface of many proteins. Carbohydrates bound to serine and threonine form O-glycosidic bonds and those linked to asparagine form N-glycosidic bonds (Perutz, 1992).
Cysteine (Cys) carries the highly reactive sulphydryl group. This does not ionise at physiological pH nor form hydrogen bonds of significant strength, but two cysteines placed some distance apart along a polypeptide chain, or forming part of different chains, can be joined by oxidation to form the disulphide bridge of cystine which plays an important part in stabilizing protein structures. Disulphide bonds increase the conformational stability mainly by constraining the unfolded conformations of the protein and thereby decreasing their conformational entropy (Pace, 1990). Cysteines also bind zinc, copper, and iron ions. The sulphur atom in methionine is unreactive and generally serves no function other than imposing a special configuration on the aliphatic sidechain, but in cytochrome c it forms the link between the protein and the heme iron (Olson, 1992).
Protein structure determination
In terms of the accuracy of protein structure determinations, all of the bond lengths are invariant. Bond angles are also essentially invariant, except perhaps for , the backbone N-Calpha-C angle. The alpha-carbon is tetrahedral, which would give 110°, but there are indications from accurately refined protein structures (Deisenhofer and Steigemann, 1975; Watenpaugh et al., 1979) that can sometimes stretch to larger values in order to accommodate other strains in the structure. The dihedral angle at the peptide is very close to 180° (producing a trans, planar peptide with the neighbouring alpha-carbons and the N, H, C, and O between them all lying in one plane). The remaining dihedral angles are the source of essentially all the interesting variability in protein conformation. The backbone dihedral angles are and in sequence order on either side of the alpha-carbon, so that is the dihedral angle around the N-Calpha bond and around the Calpha-C bond. The side chain dihedral angles are 1, 2, etc. An extremely useful device for studying protein conformation is the Ramachandran plot (Ramachandran et al., 1963) which plots and . The values of and that are possible are constrained geometrically due to steric clashes between non-neighboring atoms. The permitted values of and are indicated on a two-dimensional map of the - plane (Branden and Tooze, 1991).
Secondary Structure
The secondary structure of a segment of polypeptide chain is the local spatial arrangement of its main-chain atoms without regard to the conformation of its side chains or to its relationship with other segments (IUPAC-IUB, 1970). There are three common secondary structures in proteins, namely alpha helices, beta sheets and turns. That which cannot be classified as one of the standard three classes is usually grouped into a category called "other" or "random coil". This last designation is unfortunate as no portion of protein three dimensional structure is truly random and it is not a coil either. Regular secondary structure conformations in segments of a polypeptide chain occur when all the bond angles in that polypeptide segment are equal to each other, and all the bond angles are equal. The rotational angles for and bonds for common regular secondary structures are shown in the table below.
Max Ferdinand Perutz was born in Vienna on May 19th, 1914. Both his parents, Hugo Perutz and Dely Goldschmidt, came from families of textile manufacturers who had made their fortune in the 19th century by the introduction of mechanical spinning and weaving into the Austrian monarchy. He was sent to school at the Theresianum, a grammar school derived from an officers academy of the days of the empress Maria Theresia, and his parents suggested that he should study law in preparation for entering the family business. However, a good schoolmaster awakened his interest in chemistry, and he had no difficulty in persuading his parents to let him study the subject of his choice.
In 1932, he entered Vienna University, where he, in his own words, "wasted five semesters in an exacting course of inorganic analysis". His curiosity was aroused, however, by organic chemistry, and especially by a course of organic biochemistry, given by F. von Wessely, in which Sir F.G. Hopkins' work at Cambridge was mentioned. It was here that Perutz decided that Cambridge was the place where he wanted to work for his Ph.D. thesis. With financial help from his father he became a research student at the Cavendish Laboratory in Cambridge under J.D. Bernal in September 1936, and he has stayed at Cambridge ever since.
After Hitler's invasion in Austria and Czechoslovakia, the family business was expropriated, his parents became refugees, and his own funds were soon exhausted. He was saved by being appointed research assistant to Sir Lawrence Bragg, under a grant from the Rockefeller Foundation, on January 1st, 1939. The grant continued, with various interruptions due to the war, until 1945, when Perutz was given an Imperial Chemical Industries Research Fellowship. In October 1947, he was made head of the newly constituted Medical Research Council Unit for Molecular Biology, with J.C. Kendrew representing its entire staff. He continued holding this post until he was made Chairman of the Medical Research Council Laboratory of Molecular Biology, in March 1962. His collaboration with Sir Lawrence Bragg has continued through all these years.
The scientific work of Perutz on the structure of haemoglobin started as a result of a conversation with F. Haurowitz in Prague, in September 1937. G.S. Adair made him the first crystals of horse haemoglobin, and Bernal and I. Fankuchen showed him how to take X-ray pictures and how to interpret them. Early in 1938, Bernal, Fankuchen, and Perutz [Nature, 141 (1938) 523] published a joint paper on X-ray diffraction from crystals of haemoglobin and chymotrypsin. The chymotrypsin crystals were twinned and therefore difficult to work with, and so Perutz continued with haemoglobin. D. Keilin, then Professor of Biology and Parasitology at Cambridge, soon became interested in the work and provided Perutz and his colleagues with the biochemical laboratory facilities which they lacked at the Cavendish. Thus from 1938 until the early fifties the protein chemistry was done at Keilin's Molteno Institute and the X-ray work at the Cavendish, with Perutz busily bridging the gap between biology and physics on his bicycle. The rest of the story is well-known and forms the subject of his Nobel discourse.
Perutz has persued one sideline concerned with glaciers, studying their crystal texture and mechanism of flow, but this was mainly an excuse for working in the mountains: he is a keen mountaineer, his other recreations being walking, skiing and gardening. Scientifically, his overwhelming interest lies on the side of molecular biology. He is grateful for having had the good fortune of being joined by colleagues of great ability, several of whom have now been honoured with the Nobel Prize at the same time as Perutz himself. Kendrew came in 1946, Crick in 1948, and Watson arrived as a visitor in 1951. Recently F. Sanger, who received the Nobel Prize in 1958, also joined forces with them. Perutz is extremely happy at the generous recognition given by the Swedish Academy of Sciences and the Royal Karolinska Institute to their great common adventures and hopes that it will spur them to new endeavours.
Perutz, who is a Fellow of the Royal Society, was made Companion of the British Empire in 1962. He is also an honorary member of the American Academy of Arts and Sciences.
In 1942, Perutz married Gisela Peiser. They have two children, Vivien (b. 1944) and Robin (b. 1949).
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