Testing and improvement in our understanding of the molecular forces that influence biological phenomena (events including protein folding, protein-protein recognition, drug-receptor binding, and enzyme catalysis) require precise observations in well-controlled experiments. We developed a tool - the 'molecular torsion balance'- for measuring the relative energies of two well separated thermodynamic states which differ only in their conformation. These precision molecular tools can reliably measure differential energy effects on conformation as small as 0.05 kcal/mol. We propose to use these molecules in aqueous systems and to acquire quantitative data relevant to five important binding motifs that are central elements in theories of protein stability. The subprojects are designed to provide data directly relevant to understanding biological recognition (folding and binding) and useful for testing current biocomputational methods and theories. We will investigate: 1) The effects of salt bridges on conformational stability. Our experiments will provide quantitative comparisons of the strength of solvent-exposed salt bridges most common in biological molecules, and on the effects of ionic strength and temperature on these effects. 2) Hydrophobic binding and the effect of non-polar surfaces on water-mediated conformational stability. The Lum-Chandler-Weeks (LCW) theory of hydrophobicity makes intriguing predictions on how hydrophobicity changes with the sizes of alkyl surfaces. We seek to verify this prediction and quantify the differences. 3) The effects of halogens and the 'halogen bond'on conformation stability in water. The introduction of halogens into drug molecules is reported to change binding site affinities. 4) Neighboring group effects on hydrophobic interactions. How do nearby functional groups influence the 'stickiness'of hydrophobic surfaces? Is there an effect on the structure of water that can change the excess free energy at hydrophobic-water interfaces? 5) A beta-turn mimetic molecular torsion balance. A moderate risk -high impact subproject will allow direct comparisons of the energy of pair-wise amino acid interactions in anti-parallel orientation and the effects of amino acid changes on short beta-strand stability. Knowledge gained in these experimental studies will be available for testing current computational methods and theories of biological recognition and in identifying guiding principles for design of biologically active agents.
NIH ExPorter
NIH ExPorter