The sequence of a protein contains two types of information. At each position, each residue, represents a particular amino acid ‘nature’ and also a particular amino acid ‘identity’. Which of these is more important in causing folding to occur? We would argue that nature should be more important than identity, because noncovalent interactions (e.g., hydrogen bonding, electrostatic interactions, or van der Waals contacts including hydrophobic contacts) between amino acid residues during folding occur at such short distances that it would be foolish to assume that they determine folding – especially where long-range contacts are concerned – whereas quite clearly they do determine the stability of the folded structure, once it has been allowed to form. We would wager, therefore, that amino acid nature determines the initial folding of the polypeptide chain of a naturally-occurring protein, with amino acid identity being important only in the final stages of sidechain-sidechain packing during the very last stages of folding (because identity would determine the propensity to pack, to some extent). To test this hypothesis, we took a well-known and much-studied protein, chymotrypsin inhibitor 2 (CI2) and made variants in which almost every residue in the protein was caused to be simultaneously replaced by a residue of like nature, but different identity, using a pre-developed algorithm for introducing such replacements. We made two versions of such globally-mutated CI2 incorporating conservatively-introduced mutations, one in which valine was replaced by leucine and the other in which valine was replaced by isoleucine, all else being equal, i.e. with all other substitution mutations being made identically in both variants. We studied the proteins computationally through MD simulations, and also experimentally by making them and subjecting them to various spectroscopic analyses including NMR. We demonstrated that global conservative mutagenesis creates structural homologs of known proteins, folded similarly but not functioning similarly. The variants did not inhibit chymotrypsin.

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