Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (i) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (ii) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (iii) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.

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