Amino acid residues determined

An extensive variety of neuromuscular diseases have been uncovered as congenital conditions involving not only the nAChR and AChE but also proteins that control their expression and synaptic localization. These are discussed in Ch. 43. Study of the underlying genotype or sequence differences has also proved helpful in unraveling the involvement of various amino acid residue determinants in function [41]. 

Table III. Number of Amino Acid Residues Determined on 10% Agarose...

Cheung, E.Y., McCabe, E.E., Harris, K.D.M., Johnston, R.L., Tedesco, E., Raja, K.M.P., and Balaram, P. (2002) C-H- - -O hydrogen bond mediated chain reversal in a peptide containing a 7-amino acid residue, determined directly from powder X-ray diffraction data, Angew. Chem., Int. Ed. Engl. 41, 494-496. 

Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Heischer, S. (1999). Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor. J. Biol. Chem. 274, 15,315—15,319. 

The interactions that determine the final shape of a protein are mediated between amino acid residues in the protein. Since the sequence of amino acid residues determines the distribution of these interacting groups, the sequence encodes the overall fold of a native protein. The main interactions are as follows. 

Proteins are the most abundantly occurring natural polyampholytes. In addition to their amphoteric character, proteins are amphiphilic heteropolymers. The distribution of the monomers (the various amino acid residues) determines the amphiphi-licity and charge distribution and is therefore largely responsible for the 3D structure of a protein molecule in aqueous solution. Principles governing the protein structure and structural stability are treated in more detail in Chapter 13. 

Usually, a rapid binding step of the inhibitor I to the enzyme E leads to the formation of the initial noncovalent enzyme-inhibitor complex E-I. This is usually followed by a rate determining catalytic step, leading to the formation of a highly reactive species [E—I ]. This species can either undergo reaction with an active site amino acid residue of the enzyme to form the covalent enzyme-inhibitor adduct E—I", or be released into the medium to form product P and free active enzyme E. 

In this way each amino acid residue is associated with two conformational angles and y. Since these are the only degrees of freedom, the conformation of the whole main chain of the polypeptide is completely determined when the ([) and y angles for each amino acid are defined with high accuracy. 

Carboxypeptidases are zinc-containing enzymes that catalyze the hydrolysis of polypeptides at the C-terminal peptide bond. The bovine enzyme form A is a monomeric protein comprising 307 amino acid residues. The structure was determined in the laboratory of William Lipscomb, Harvard University, in 1970 and later refined to 1.5 A resolution. Biochemical and x-ray studies have shown that the zinc atom is essential for catalysis by binding to the carbonyl oxygen of the substrate. This binding weakens the C =0 bond by... 

There are at least three different classes of crystallins. The a and (3 are heterogeneous assemblies of different subunits specified by different genes, whereas the gamma (y) crystallins are monomeric proteins with a polypeptide chain of around 170 amino acid residues. The structure of one such Y crystallin was determined in the laboratory of Tom Blundell in London to 1.9 A resolution. A picture of this molecule generated from a graphics display is shown in Figure 5.11. 

Many biochemical and biophysical studies of CAP-DNA complexes in solution have demonstrated that CAP induces a sharp bend in DNA upon binding. This was confirmed when the group of Thomas Steitz at Yale University determined the crystal structure of cyclic AMP-DNA complex to 3 A resolution. The CAP molecule comprises two identical polypeptide chains of 209 amino acid residues (Figure 8.24). Each chain is folded into two domains that have separate functions (Figure 8.24b). The larger N-terminal domain binds the allosteric effector molecule, cyclic AMP, and provides all the subunit interactions that form the dimer. The C-terminal domain contains the helix-tum-helix motif that binds DNA. 

The simple lesson to be learnt from these experiments is that critical amino acid residues can have pleiotropic roles in determining a protein s structure and therefore its function. 

The molecular basis for quasi-equivalent packing was revealed by the very first structure determination to high resolution of a spherical virus, tomato bushy stunt virus. The structure of this T = 3 virus was determined to 2.9 A resolution in 1978 by Stephen Harrison and co-workers at Harvard University. The virus shell contains 180 chemically identical polypeptide chains, each of 386 amino acid residues. Each polypeptide chain folds into distinct modules an internal domain R that is disordered in the structure, a region (a) that connects R with the S domain that forms the viral shell, and, finally, a domain P that projects out from the surface. The S and P domains are joined by a hinge region (Figure 16.8). 

The specific role of each amino acid residue for the function of the protein can be tested by making specific mutations of the residue in question and examining the properties of the mutant protein. By combining in this way functional studies in solution, site-directed mutagenesis by recombinant DNA techniques, and three-dimensional structure determination, we are now in a position to gain fresh insights into the way protein molecules work. 

Sanger also determined the sequence of the A chain and identified the cysteine residues involved in disulfide bonds between the A and B chains as well as in the disulfide linkage within the A chain. The complete insulin structure is shown in Figure 27.11. The structure shown is that of bovine insulin (from cattle). The A chains of human insulin and bovine insulin differ in only two amino acid residues then B chains are identical except for the amino acid at the C terminus. 

Whereas the primary structure of a protein is determined by the covalently linked amino acid residues in the polypeptide backbone, secondary and higher... 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

The structure of the UQ-cyt c reductase, also known as the cytochrome bc complex, has been determined by Johann Deisenhofer and his colleagues. (Deisenhofer was a co-recipient of the Nobel Prize in Chemistry for his work on the structure of a photosynthetic reaction center [see Chapter 22]). The complex is a dimer, with each monomer consisting of 11 protein subunits and 2165 amino acid residues (monomer mass, 248 kD). The dimeric structure is pear-shaped and consists of a large domain that extends 75 A into the mito-... 

The specific ribonucleotide sequence in mRNA forms a message that determines the order in which amino acid residues are to be joined. Each "word," or codon, along the mRNA chain consists of a sequence of three ribonucleotides that is specific for a given amino add. For example, the series UUC on mRNA is a codon directing incorporation of the amino acid phenylalanine into the growing protein. Of the 43 = 64 possible triplets of the four bases in RNA, 61 code for specific amino acids and 3 code for chain termination, fable 28.1 shows the meaning of each codon. 

Glycine receptor function is modulated by alcohols and anesthetics [4]. Amino acid residue al(S267) is critical for alcohol potentiation, as mutation to small residues (Gly, Ala) enhance, and mutation to large residues (His, Cys, Tyr) diminish the ethanol effect. Glycine recqrtor modulation by Zn2+ involves structural determinants located within the large N-terminal domain. Additional glycinergic modulators include neuroactive steroids and the anthelmintic, ivermectin, which activates glycine receptors by a novel, strychnine-insensitive mechanism. 

Sequence of amino acids that determine the transport of proteins into the nucleus. Although there is no clear consensus, nuclear localization signals tend to be rich in positively charged residues, which allow interaction with proteins from the nuclear import machinery (i.e., importins). 

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