PROBING PROTEIN STABILITY WITH NONNATURAL AMINO-ACIDS

Quantitative replacement of methionine with its non-natural amino acid analogues, norleucine, selenomethionine and telluro-methionine in hum an recombinant annexin V, is applied to study conformational and foldi ng properties in solution. This procedure replaces each methionine sul phur atom with Se, Te or -CH2-, providing single-atom exchanges, or >> atomic mutations<<. Using guanidine chloride as denaturant, the estima ted stabilities of protein variants are not significantly changed. The denaturation midpoints are shifted towards lower values owing to the increase in the hydrophobicity of exchanged residues. Co-operativity e xpressed in terms of m-values is also affected by such exchanges and i s highly correlated with the physical properties of methionine and its analogues in solution. This approach can contribute to a detailed und erstanding of interactions of particular amino acids and their implica tions on protein folding and protein-ligand interactions.