Protein and peptide structure and interactions studied by hydrogen exchanger and NMR

Hydrogen exchange is widely used, especially in the study of protein stability and dynamics, folding, and interactions. Most applications require quenching of the exchange. Some of the methods have limitations due to insufficient quench. We studied the quenching properties of solvent mixtures. The pH dependence of the exchange rates in DMSO/D$\sb2$O mixtures is the same as that in water, but with suppressed minimum rate, k$\sb{\rm min}$, and elevated optimal pH. The decrease of k$\sb{\rm min}$ is due to the dramatic reduction of the base catalyzed exchange caused mainly by the changes in the relevant pKa\u27s. By solvent quenching, it is possible to measure exchange rates for peptides in situations where direct NMR measurement is not applicable due to overlap in 1D and exchange during 2D experiments. This new approach was used to measure the exchange rates of monomeric melittin in aqueous solution and when bound to calmodulin. Our results indicate that the C-terminal half of melittin binds calmodulin more tightly then the N-terminal half. The protection pattern is consistent with bound melittin being $\alpha$-helical. The new method allows more detailed mapping of the binding sites involved in protein-protein interactions since one can now observe the protected amide protons on protein surface, which exchange rapidly in the free protein. A synthetic peptide (CHIGPGRAFC), derived from the V3 region of gp120 of HIV-1, in aqueous solution was investigated by NMR. The spectrum of the reduced peptide is characteristic of an unstructured, highly flexible coil. Disulfide bond cyclization results in major changes in chemical shifts and gives rise to NOE\u27s indicative of well-defined turns. The presence of these turns is supported by the low temperature coefficients of the chemical shift of the amide protons of Arg7 and Phe9. The conformation of the peptide is in good agreement with that reported for this sequence as part of a longer peptide bound to an HIV neutralizing antibody, which might be similar to that of the corresponding fragment on the surface of the intact virus. The artificially introduced disulfide bond appears to reinforce the inherent structural tendencies