Chapter 2 Section 3 PROTEINS
2. Secondary structure refers to local conformations formed by neighbouring amino acids in polypeptide chains. These patterns are repeated in diverse proteins and are enabled by extensive
H-bonding between parts of the same or different polypeptide chains. Prevalent secondary structures are the α-helix, β-pleated sheet and β-turns. In contrast, those protein conformations that do not exhibit a repeated pattern are called In contrast, those protein conformations that do not exhibit a repeated pattern are called random coils.
The α-helix is a tightly-coiled, right-handed spiral formed by the peptide chain. Each amino acid in the backbone is twisted equally. The spiral ascends 1.5 Å along the longitudinal axis between one amino acid and the next one in the chain. A single complete turn of the helix is a repeat unit;
it is composed of 3.6 amino acid residues and ascends 5.4 Å along the longitudinal axis. Every -Cα-Cβ bond (i.e. R group) projects out in a direction nearly perpendicular to the main axis.
c. View down axis of helix d. Space-filling model of a part of helix
Fig 2.3.20. Schematic representations of the α-helix are shown in (a-c); (d) is a part of an α-helix showing proximity of side-chains of Asp100 (red) and Arg 103 (blue) residues which attract each otherelectrostatically
(Source: (a-c) Murray et al, 2003, p 32 figs 5-2 and 5-3; p33 fig 5-4 (d) Nelson and Cox, 2005, p 121 fig 4-5)
Each turn of the helix is held to the adjacent turns by 3-4 intramolecular H-bonds, running parallel to the main axis. They are formed between the amide hydrogen in the peptide bond of one amino acid, and the carbonyl oxygen in the peptide bond of an amino acid which is 3 residues away from it in the chain. The H-bonds hold the coils together, making the structure stable, and yet flexible and elastic. Many α-helices are amphipathic since they tend to have hydrophobic R groups on one side of the axis and hydrophilic ones on the other side. Hence they can form interfaces between the hydrophobic interior of a protein and the aqueous medium.
The α-keratin molecule in hair and nail, and elastin in connective tissue are examples of α helical structures. The “coiled coil” of collagen is a special helical secondary structure and is illustrated below:
A. B.
Fig 2.3.21. Schematic diagrams of collagen:
(A) shows the typical triple-helix structure in a part of a collagen molecule. Each strand is a left- handed helix and three strands are twisted around each other to form a right handed super-helix
(Source: Lodish et al, 2003, p211 fig 6-14)
(B) is a part of a collagen fibril showing the cross-linking between collagen molecules that imparts tensile strength. (Source: Nelson and Cox, 2005, p 128 fig 4-13)
Since the peptide bond is a dipole, the presence of other positively charged side-chains near the C- terminal end, and negatively charged side-chains near the N-terminal end, stabilizes the coiled structure.
Hydrophobic and van der Waal’s interactions at the core stabilize the helix. Proline and Gly disrupt the helix, producing bends. Mutual repulsion between identically charged amino acids (e.g. long segment with only Glu residues) or physical interference between bulky R groups destabilizes the helix.
β-pleated sheet is formed by parallel, zig-zag polypeptide chains, held together by intermolecular H-bonds at right angles to their length. The sheet is pleated at right angles to its average plane because successive carbon atoms are alternately slightly above and below the plane of the paper.
Each amino acid is rotated by 180o relative to its adjacent residues, about an axis parallel to the direction of the chain. Hence adjacent ‘R’ groups extend in opposite directions above and below the plane. The polypeptide chains can be parallel or anti-parallel with regard to the amino-to- carboxyl orientation.
Anti-parallel chains Parallel chains
Fig 2.3.22. β-pleated sheet showing parallel and anti-parallel chains (blue dots represent the α-amino groups which donate H for forming H-bonds (Source: Murray et al, 2003, p 33 fig 5-5)
Fig 2.3.23. β-pleated sheet structure of silk fibroin. (Source: Nelson and Cox, 2005, p 129 fig 4-14)
The stability of the β-sheet is primarily due to H-bonds between segments of the same or different polypeptide chains. The structure is not elastic since the polypeptide chains are fully extended.
The β-turns/bends/loops are connecting links between successive/adjacent lengths of α-helix or β-conformations. They are found more frequently in globular proteins, where the polypeptide
Fig 2.3.24. A β-turn between two anti-parallel chains (blue) in a segment of a β sheet.
(Source: Murray et al, 2003, p 34 fig 5-7)
A supersecondary structure or motif is a stable, geometrical arrangement of secondary structures.
Such clusters may occur many times in the same protein and are often associated with specific functions. Several hundred motifs are known and the same motif may be a part of many diverse proteins. One of the simplest motifs is the α-α motif which is associated with the ability to bind to DNA or to sequester a calcium ion. The β-α-β motif has 2 consecutive parallel strands of a β- sheet, connected by an α-helix. The α/β barrel motif found in many enzymes, has repetitive β-α-β loops arranged to resemble a barrel (see also triose phosphate isomerase in fig.2.3.26); it often has a “pocket” to bind a co-factor or a substrate.
α-α corner β-α-β loop β-barrel Fig 2.3.25. Common motifs in proteins (Source: Nelson and Cox, 2005, p 140 fig 4-20)
Secondary structures may assemble to form domain(s) in certain regions of a polypeptide chain.
A domain is a particularly stable section of protein structure which can fold independently and may perform a specific task (e.g. anchorage, ligand-binding).
A. Four similar protein domains B. Separate functions of domains in a CD4 molecule in an IgG antibody Fig 2.3.26. Domain structures in proteins shown by a ‘ribbon diagram” in (A) and by a schematic figure in (B).
(Source: Berg et al, 2002, fig 4.30)
3. Tertiary structure is the 3-D, thermodynamically stable structure of the protein. Amino acids that may be far apart in 1o structure and are constituents of different 2o structures, interact by non- covalent forces and disulfide bonds to give the native, folded conformation of the protein.
Hydrogen and ionic bonds, and hydrophobic forces determine tertiary structure. Hydrophobic side-chains are forced into the interior of the protein molecule where their interaction with water is minimized and the van der Waal’s binding of hydrophobic groups is maximized.
For some proteins, the complex molecule formed by the 1o, 2o and 3o structures may be the functional protein. However, in others it may need association with a complementary molecule(s) in order to attain the functional state.
4. Quarternary structure is exhibited by those proteins in which the final functional molecule is produced by the fitting together of separate, coiled and folded sub-units. The constituent polypeptide chains may be identical (e.g. phosphorylase a) or different (e.g. globin in Hb). Non- protein components may also be present e.g. ribosomes (protein sub-units + RNA). Multimeric proteins which have identical sub-units or repeating groups in non-identical subunits are generally symmetrical. Symmetry may be rotational/helical, giving more closed/open-ended structures respectively.
Folding of proteins in vivo is a very rapid process. It may be spontaneous or aided by molecular chaperones. Failure of proper folding causes serious neurologic disorders like Alzheimer’s disease, bovine spongiform encephalopathy (mad cow disease) and other prion diseases.
Proteins may also show relatively disordered regions of high conformational flexibility, which become ordered on binding of a ligand. Such regions are useful in enzymes which bind substrates by the induced-fit method.
Depicting the conformation of protein structures
The Protein Data Bank (PDB) with its easy access, is a very helpful tool for biochemists. The amino acid sequences of numerous proteins are listed in detail in protein databases.
VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTN AVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTS KYR
Fig 2.3.27. Representation of the amino acid sequence in the α-chain of human hemoglobin.
Matching of amino acid sequences of different proteins are done to identify and compare them on the basis of their primary structure. However, such amino acid sequences are often lengthy and the functional role of a protein is not always understandable. Many types of simple graphic models are now used to depict tertiary and quarternary structures, so that the conformation of a protein molecule is easy to comprehend. Dynamic computer-aided simulations from these static diagrams help to envisage ligand-binding and rotational abilities of the proteins.
• “Mesh” representation – emphasizes the protein surface
• Solvent accessibility image – depicts hydrophilic and hydrophobic regions on the surface
• Surface contour image – visualizes pockets where ligands can be bound
• Space-filling models – each atom is shown as a sphere encompassing its van der Waal’s radius. All side chains are shown.
• Ball-and-stick model – shows the molecular constituents
Fig 2.3.28. Ribbon diagram of triose phosphate isomerase (Source: Murray et al, 2003, p 34 fig 5-6)
Ribbon diagram “Mesh” image Surface contour image Space-filling model Fig 2.3.29. Different modes of representation of the 3-D structure of a protein viz. sperm whale myoglobin.
(Source: Nelson and Cox, 2005, p 133 fig 4-16)
Ball-and-stick model Solvent accessible model (regions of positive charge in blue and negative charge in red)
Fig 2.3.30. Different models of the 3-D structure of Ras, a GTP-binding protein (Source: (Lodish et al, 2003, p 63 fig 3-5)
The complete content of protein databases has been organized to group proteins into four classes on the basis of their structural motifs:
• all α e.g. serum albumin,
• all β e.g. Immunoglobin–like sandwich,
• α/β e.g. phosphofructokinase, and
• α and β e.g. ubiquitin conjugating enzyme.
This grouping is called the structural classification of proteins (SCOP). The SCOP database helps to identify conserved protein structures in evolution. Proteins with significant similarity in primary sequence or demonstrable similarity in structure and function are grouped into one family. Two or more families with little similarity in primary sequence but with similarity in some major motif and function, are clubbed together in the same super-family e.g. several cell adhesion molecules with immunoglobulin-like domains are grouped under same Ig superfamily.
2.3.5. PROTEOMICS
The new field of proteomics is the collective study of the entire complement of proteins in an organelle/cell/organism. It differs from conventional protein chemistry by dealing with large numbers of proteins at a time. Unlike the genome which is identical in all somatic cells, the proteome varies from cell to cell and from time to time as per requirements and responses of the organism to its internal and external environment.
Since the synthesis of proteins is strictly determined by genes, proteomics indirectly and reliably, gives us a clear idea of the functioning of the genome in cells. Modern analytical procedures like mass spectrometry together with computational search of protein and DNA databases, enable proteomics and genomics to be correlated. Evolutionary homologies have been understood and applied to biotechnology. The result is a spectacular leap in measures related to crop improvement, clinical diagnostics (using protein biomarkers), drug designing, and medical intervention in the treatment of disease. An international collaboration has been undertaken by the Human Proteome Organisation to catalogue all proteins and their functions and interactions.