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To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, we have constructed a series of variant a subunits of tryptophan synthase from Escherichia coli substituted by each of 20 amino acids at position 49, which is buried in the interior of the protein. The stabilities were quantitatively examined except for the mutant protein substituted by arginine, which was not obtained in enough quantity. The Gibbs energy of unfolding in water and the activation Gibbs energy of unfolding in 3 M guanidine hydrochloride for each protein were compared at pH 7.0 and pH 9.0. The Gibbs energy of unfolding in water at pH 7.0 varied from 0.72 to 1.92 times that of the wild-type protein by the substitutions, but the activation Gibbs energy of unfolding in 3 M guanidine hydrochloride varied only from 0.95 to 1.03 times that of the wild-type protein. Moreover, the stability of the protein substituted at this position, which is buried in the interior of the molecule, tended to increase linearly with increasing hydrophobicity of the substituted residue, unless the volume of the substituted residue was over a certain limit.We still cannot fully predict the three-dimensional structure of a protein on the basis of the amino acid sequence, although the sequence determines the three-dimensional structure of the protein under physiological conditions. On the other hand, recent advances in molecular biology have given us the ability to modify the gene of a protein virtually without limitations (1). This shows that if we can predict the threedimensional structure from the amino acid sequence, it will help greatly in designing a desired protein. To do so, however, more information is needed on the folding mechanism and stabilization of three-dimensional structure and their relationship to amino acid sequence.The study of the effect of single amino acid substitutions on conformational stability using mutant proteins is a fruitful approach to understanding the role of the amino acid sequence in protein folding and protein stability. The stabilities of mutant forms of tryptophan synthase ct subunit (2-6) and T4 phage lysozyme (7) have been studied quantitatively. In the case of tryptophan synthase a subunit from Escherichia coli, the conformational stabilities of wild-type and of six mutant proteins substituted at position 49, which is buried in the interior of the protein, have been compared. The results indicate that (i) the proteins easily become more stable or less stable to denaturant as a result of single amino acid substitutions and (ii) the stabilities of the proteins tend to increase linearly with hydrophobicity of the substituting residues (8).It is widely appreciated that the hydrophobic interaction is important in protein folding and protein stability (9-11).However, the role of hydrophobic interaction in the conformational stability of a protein has yet to be quantitatively evaluated for each amino acid residue. The purpose of this article is to show how the hydrophobicity of the su...

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