THERMODYNAMICS OF HYDROPHOBIC INTERACTIONS ACROSS A PROTEIN BETA-SHEET

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Interactions between amino acids are crucial for protein folding. To investigate such interactions, a zinc finger-based host-guest system is used. Folded single TFIIIA-like zinc finger peptides contain an anti-parallel b-sheet and an a-helix but are unfolded in the absence of metal. The host is a model single TFIIIA-like zinc finger peptide, and the guests are valines in two solvent exposed non-hydrogen bonded lateral pairings within the beta-sheet.Absorption spectroscopy cotitrations were performed to monitor metal binding. An internal standard peptide was included for a competitive assay to maximize precision in metal ion dissociation constant determination. Valines substituted for serine in the two positions had different effects on the deduced folding standard free energy. There is no excess Val-Val DDG¿interaction, and thus contributions from each valine to DDG¿interaction are additive.Single peptide, single metal titrations using ITC were used to determine the Val-Val DDH¿interaction and to calculate DDS¿interaction. DDH¿interaction is moderately endothermic and DDS¿interaction is favorable. Enthalpic-entropic compensation occurs to achieve zero DDG¿interaction. The release of water is apparently important and gives the favorable entropic contribution. These thermodynamic results are being compared to the structure of the Val-Val peptide, using NMR of the Zn and Co complexes. The structure of the peptide-Zn complex determined using conventional 2D techniques resulted in a structure without enough constraints to access the interaction between the valine pair. Determination of the paramagnetic susceptibility and residual dipolar coupling tensors of the peptide-Co complex will be used to provide further constraints. The determination of the paramagnetic susceptibility tensor will lead to the addition of constraints from paramagnetic pseudo-dipolar chemical shifts and residual dipolar coupling of the molecule aligned in different fields.