Conservation change

Now we can ask what is likely to happen to the three-dimensional structure of a protein if we make a conservative replacement of one amino acid for another in the primary structnre. A conservative replacement involves, for example, substitution of one nonpolar amino acid for another, or replacement of one charged amino acid for another. Intnitively, one would expect that conservative replacements would have rather little effect on three-dimensional protein structure. If an isoleucine is replaced by a valine or leucine, the structnral modification is modest. The side chains of all of these amino acids are hydrophobic and will be content to sit in the molecnlar interior. This expectation is borne out in practice. We have noted earlier that there are many different molecnles of cytochrome c in nature, all of which serve the same basic function and all of which have similar three-dimensional structnres. We have also noted the species specificity of insulins among mammalian species. Here too we find a number of conservative changes in the primary structure of the hormone. Although there are exceptions, as a general rule conservative changes in the primary structnre of proteins are consistent with maintenance of the three-dimensional structures of proteins and the associated biological functions. 

Molecular modelling of butadiene oxidation by CYP2E1 has indicated that species differences in the kinetic parameters might be explained by a non-conservative change from Thr-437 to His-437 between rodents and humans and by a conservative change from Ile-438 to Val-438 (Lewis etal., 1997). 

For many years patients with diabetes were treated with insulins that had been isolated from the pancreases of pigs and cows. The primary sequences of these insulins are closely related to the sequence of human insulin. There is only a single difference between the sequences of human and porcine insulins human insulin has a threonine at position B30, and porcine insulin has an alanine. Bovine insulin differs from human insulin at three positions. There is an alanine at the B30 position, an alanine at A8, and a valine at A10. These conservative changes in primary sequence have no apparent effect on biologic activity, although there are slight differences in solubility (7). 

It is worth noting that a conservative change from Glu to Asp resulted in higher specific activities in all three types of assays. It is interesting that all higher plant branching-enzymes have an Asp at the position occupied by Glu-459 in E. coli. 

E. coli lipoamide dehydrogenase has a valine residue preceding the first half-cystine, whereas the eukaryote enzymes have a threonine residue. Chemically this is a relatively conservative change since the side chains of these amino acids are virtually identical in volume. 

We are not suggesting that the aforementioned considerations are always critical. Generally, the structure and function of a domain are not altered by conservative point mutations or even by removing parts of the sequence not involved in the domain. However, there is always the chance that even a conservative change in the sequence can have a dramatic impact. Thus, the more the structure differs from the full-length target sequence, the less certain the conclusions that you can draw from it. 

Figure 5 Multiple sequence alignment of carboxypeptidases. Similarity of the enzymatically active subunit of human carboxypeptidase N (7) to carboxypeptidase A from bovine (1), rat (3), human mast cell (4), bovine carboxypeptidase B (2), human carboxypeptidase M (5), and bovine carboxypeptidase H (6). Residues identical and conservative changes in at least four proteins are boxed. Arrows indicate active site residues. (From N Refs. 119-123.)... 

One eleven-amino acid segment of chain, from 70 to 80, was thought for many years to be absolutely essential because it was invariant among all species then known. More recently this invariance has been broken, but in a conservative manner. Tyrosine-74 has been observed as phenylalanine in Humicola and Crithidia, and isoleucine-75 is valine in Euglena and methionine in Crithidia. (These two species also have the anomalous alanine at position 14.) Such conservative changes in a small number of distantly related eukaryotic organisms do not detract from the prediction that the left side of the molecule, residues 70-80, must have an important functional role that keeps its sequence so nearly invariant. 

To date, more than 60 members of the A-type ASTs are structurally known from insects and about 20 species have been analyzed. Although only three species of decapod crustaceans were investigated, more than 60 isoforms are known in these crustaceans (see Table 4) interestingly, none is identical to the ASTs identified so far in insects [139]. Thusfar, in each insect and crustacean species investigated to date, multiple isoforms of the A-type Asts have been isolated or demonstrated to be present (see Table 4). Whereas all members of the A-type Ast family contain the relatively conserved pentapeptide core C-terminal of Y/FXFGLamide (for Leu there can be conservative changes such as lie or Val but also Met in the blowfly, Calliphora vomitoria), the N-terminal part of the peptide can... 

The point should be made before detailed considerations of site-directed mutagenesis, etc., that changes in particular residues of P450 2C9 yield markedly different effects depending on the substrate and reaction under consideration. For instance, the polymorphism 3 (I359L), which appears to be very conservative, changed catalytic efficiencies of different reactions by factors of... 

An early study of the cytochrome c protein structure revealed that some portions of the surface seemed to be experiencing unusually high functional constraint [72]. These surface residues were determined to be sites of interaction with other proteins (interfaces). Subsequent studies have generally supported the notion that interfacial surfaces are more conserved than the remainder of the protein s solvent-exposed surface, and slightly less conserved than the protein s core [73]. Substitutions that do occur in the interface are heavily skewed toward more conservative changes [53], as defined by the Grantham classification scheme [74]. Exploiting the difference in evolutionary rate between interfacial and non-interfacial sections of a protein s surface has been proposed as a means by which to identify interfaces in newly characterized proteins this has proven to be difficult in practice [75]. 

We also note that the change in metal ion specificity has been accomplished exclusively via the use of subtle changes in the positions of second sphere residues, or very conservative changes in their identities. This is consistent with the constraint that the evolutionary intermediates could not lack SOD activity altogether if the carrier organism were to survive. 

Maitotoxin causes two highly conserved changes in a wide variety of cells. It induces a rapid and sustained elevation of [Ca i and Na ([Na ]i), and as a result, a strong depolarization. The influx of Ca and Na induced by MTX appears to involve the activation of Ca -permeable, nonselec-tive cation channels (CaNSC). Simultaneous electrophysiological and fluorescent measurements of [Ca ]i and [Na ]i upon MTX-CaNSC activation reveal similar kinetics indicating that this channel is responsible for the flux of both ions. 

Figure 3. Comparison of aligned amino acid sequences in the PEST-like region and the cleavage domain of 32K from Spirodela oligorrhta (Avni et al, unpublished) to the L-subunit of Rhodobactor sphaeroides (5). Alignment adapted from refs. 3-5 and 9. Identical residues. Conservative changes.

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