The contribution of tyrosine water=hydrogen bonds to protein stability

Abstract

The goal of this research was to determine how protein stability is affected when tyrosines form specific inter and/or intramolecular hydrogen bonds in the folded state. Our model protein, the enzyme RNase Sa, contains four Tyr residues believed to form one or more intermolecular hydrogen bonds to surface or partially buried water molecules. To study these interactions the single mutants Tyr 30, 49, 55, 81 ʾPhe were prepared and their conformational stability and thermodynamics of folding analyzed. From thermal denaturation data the free energy of unfolding, DGu, enthalpy of unfolding, DH, the melting temperature, Tm, and heat capacity change associated with unfolding, DCp, have been calculated. Initial analysis of Tyr 30, 49, 55, 81 predicted that each residue formed hydrogen bonds to one or more water molecules; however, thermodynamic and NMR data from this study support the surprising results that Tyr 81 actually makes an atypical intramolecular bond contributing 1.2 KcaI/mol to stability. Data for Tyr 30, 49, 55 support the prediction that intermolecular hydrogen bonds to water molecules are formed.