THERMODYNAMICS OF HYDROPHOBIC INTERACTIONS ACROSS A PROTEIN BETA-SHEET

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。氨基酸之间的相互作用对蛋白质折叠至关重要。为了研究这种相互作用，使用基于锌指的主-客体系统。折叠的单个TFIIIA样锌指肽含有反平行的b-折叠和a-螺旋，但在不存在金属的情况下未折叠。主体是一个模型单TFIIIA样锌指肽，客体是两个暴露于溶剂中的β-折叠内的非氢键横向配对的缬氨酸。包括用于竞争性测定的内标肽，以最大化金属离子解离常数测定的精确度。缬氨酸取代丝氨酸在这两个位置有不同的影响推导的折叠标准自由能。不存在过量的Val-Val DDG <$相互作用，因此每个缬氨酸与DDG <$相互作用的贡献是加和的。使用ITC的单肽、单金属滴定法来确定Val-Val DDH <$相互作用并计算DDS <$相互作用。DDH相互作用是适度吸热的，DDS相互作用是有利的。发生焓-熵补偿以实现零DDG相互作用。水的释放显然是重要的，并给出了有利的熵贡献。这些热力学结果被比较的Val-Val肽的结构，使用NMR的Zn和Co络合物。使用常规2D技术确定的肽-Zn复合物的结构导致没有足够的约束来访问缬氨酸对之间的相互作用的结构。肽-Co复合物的顺磁磁化率和残余偶极耦合张量的测定将用于提供进一步的约束。顺磁磁化率张量的确定将导致从顺磁伪偶极化学位移和在不同场中排列的分子的残余偶极耦合的约束的添加。

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.