Defining the role of conformational entropy in high affinity protein interactions
定义构象熵在高亲和力蛋白质相互作用中的作用
基本信息
- 批准号:9191582
- 负责人:
- 金额:$ 5.43万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2016
- 资助国家:美国
- 起止时间:2016-09-01 至 2018-08-31
- 项目状态:已结题
- 来源:
- 关键词:AffinityAntitoxinsBacillus amyloliquefaciens ribonucleaseBindingBinding ProteinsBiological AssayCalorimetryCell physiologyComplexDataDiseaseDissociationDistantDisulfidesDrug DesignEntropyEventFinancial compensationFree EnergyGoalsHydrophobic InteractionsKineticsLaboratoriesLeadLigandsLiteratureMeasurementMeasuresMethodsMicellesMolecularMotionNMR SpectroscopyPriceProtein DynamicsProteinsProxyRelaxationReportingResidual stateResourcesRoleSideSolventsStructureSystemTechnologyTestingThermodynamicsTimeToxinVertebral columnWaterWorkbasecolicinenthalpyinhibitor/antagonistinterestinterfacialmeternovelnovel strategiesprotein complexprotein protein interactiontool
项目摘要
Project Summary
Protein binding events are essential to most cell functions and often lead to disease when disrupted. Of
particular interest here are very high affinity protein interactions with dissociation constants (Kd) in the
femtomolar range. The physical origin of the large binding free energy involved in these interactions is not well
understood. In the literature, these extreme affinities have been largely ascribed to enthalpic contributions
(structural interactions) and the hydrophobic effect (entropy of water). In preliminary results with
barnase:barstar, one of the highest affinity protein-protein interactions known, we found that this view is
incomplete. Recently, the Wand laboratory has developed a “conformational entropy meter” that is able to
quantitatively relate measurements of fast (ps-ns) dynamics of methyl-bearing side chains to the
conformational entropy. Conceptually, the approach relies on the idea that the motion indirectly reports on the
distribution of microstates accessible to the system. Our initial study of the high affinity barnase:barstar
complex reveals an unprecedented role for conformational entropy. A widespread increase in fast motions
occurs upon formation of the complex, particularly in regions distant from the binding interface. This
corresponds to a large and favorable change in conformational entropy upon binding of about -18 kcal/mol. It
follows that we should be able to decrease the binding affinity of extremely high affinity complexes by
restricting motions in barnase:barstar. To test this, intramolecular disulfide bridges will be introduced in both
proteins to rigidify the structures (akin to molecular stapling). The effect on global thermodynamics will be
studied by calorimetry. Affinties in the very high regime will be measured by a competitive inhibition kinetics
assay. Successful candidates with decreased affinity will be studied using NMR relaxation methods to measure
dynamics of the backbone and methyl-bearing sides chains. The “conformational entropy meter” will be used to
interpret quantitatively the changes in motion as changes in TΔSconf. Furthermore, the generality of this
approach will be evaluated by using the same strategy to study other extreme affinity complexes, such as the
bacterial cognate protein complex E9:Im9 (Kd ~ 10 M). Lastly, the total binding entropy of barnase:barstar
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has been reported as near-zero. This means that the contribution we find of -18 kcal/mol from TΔSconf must be
compensated by a similar but unfavorable contribution from solvent entropy. This is consistent with the ~20
water molecules seen trapped at the interface in the crystal structure. To test whether these waters exist in
solution and are truly constrained, reverse micelle technology will be used to trap single proteins with only a
few layers of water. Confinement in reverse micelles allows tracking of the protein-water interaction times and
will reveal the slowed dynamics of water molecules trapped at the barnase:barstar interface. This work will
directly evaluate the role of conformational entropy in the formation of very high affinity protein complexes.
项目概要
蛋白质结合事件对于大多数细胞功能至关重要,并且当被破坏时通常会导致疾病。
这里特别感兴趣的是非常高亲和力的蛋白质与解离常数(Kd)的相互作用
飞摩尔范围内涉及这些相互作用的大结合自由能的物理起源尚不清楚。
在文献中,这些极端的亲和力很大程度上归因于热函的贡献。
(结构相互作用)和疏水效应(水的熵)。
barnase:barstar,已知亲和力最高的蛋白质-蛋白质相互作用之一,我们发现这种观点是
最近,Wand实验室开发出了一种“构象熵计”,能够
甲基侧链快速(ps-ns)动力学的定量相关测量
从概念上讲,该方法依赖于运动间接报告构象熵的想法。
我们对高亲和力 barnase:barstar 的初步研究。
复合体揭示了构象熵的前所未有的作用,快速运动的广泛增加。
发生在复合物形成时,特别是在远离结合界面的情况下。
它对应于结合后构象熵的巨大且有利的变化,约为-18 kcal/mol。
由此可见,我们应该能够通过以下方式降低极高亲和力复合物的结合亲和力
为了测试这一点,将在两者中引入分子内二硫键。
蛋白质使结构僵化(类似于分子装订)对整体热力学的影响将是
通过量热法研究非常高的亲和力将通过竞争性抑制动力学来测量。
亲和力降低的成功候选者将使用 NMR 弛豫方法进行研究以进行测量。
主链和带有甲基的侧链的动力学将被用来测量。
将定量运动的变化解释为 TΔSconf 的变化。此外,这具有普遍性。
该方法将通过使用相同的策略来研究其他极端亲和力复合物进行评估,例如
细菌同源蛋白复合物 E9:Im9 (Kd ~ 10 M) 最后,barnase:barstar 的总结合熵。
-15
据报告接近于零,这意味着我们发现 TΔSconf 的贡献一定是 -18 kcal/mol。
由溶剂熵的类似但不利的贡献补偿,这与〜20一致。
水分子被困在晶体结构的界面上,以测试这些水是否存在于晶体结构中。
解决方案并且确实受到限制,反胶束技术将用于仅用
反胶束中的几层水可以跟踪蛋白质-水相互作用的时间和
这项工作将揭示被困在 barnase:barstar 界面的水分子的减慢动力学。
直接评估构象熵在极高亲和力蛋白复合物形成中的作用。
项目成果
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{{ truncateString('JOSE A CARO', 18)}}的其他基金
Defining the role of conformational entropy in high affinity protein interactions
定义构象熵在高亲和力蛋白质相互作用中的作用
- 批准号:
9402239 - 财政年份:2016
- 资助金额:
$ 5.43万 - 项目类别:
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