CONFORMATIONAL STABILITY OF GLOBULAR PROTEINS

Texas A&M University Health Science Ctr, College Station TX

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Abstract

Proteins can now be constructed with any amino acid sequence. The importance of applications of this technology in health and other areas is now clear and limited mostly by our current knowledge and imagination. Thus, it is essential that we learn to predict how changes in the amino acid sequence will affect the function, folding, and stability of a protein. The primary goal of this project is to gain a better understanding of the forces that contribute to the conformational stability of globular proteins and to use this information to develop methods to increase their stability. This requires a thorough understanding of the structures of the folded and unfolded conformations of proteins and of the effect of changes in structure on the equilibrium between these states. To this end, we will continue to study the effect of small changes in the amino acid sequence on the conformations of the folded and unfolded states, and on the thermodynamics of folding of five microbial ribonucleases: RNase Sa, RNase Sa2, RNase Sa3, RNase T1, and RNase Ba, also known as barnase. Some of the questions we hope to answer are: 1) Can mutations such as Phe yields Tyr that add hydrogen bonds to folded proteins be used to increase their stability? 2) Can mutations such as Gly yields Thr that both reduce the conformational entropy of the unfolded state and bury nonpolar surface area or form hydrogen bonds in the folded state be used to increase protein stability? 3) Can an I to I+4 interaction between glutamine and aspartate side chains on the exposed face of an alpha- helix be used to increase protein stability? 4) Can we double the conformational stability of ab RNase without reducing the enzymic activity? 5) How does the net charge on a protein effect its conformational stability and other physical and chemical properties? 6) What determines the pK values of the ionizable groups in folded and unfolded proteins and how do they effect the conformational stability?