INTERPRETING CONFORMATIONAL DIFFERENCES IN THE ALLOSTERIC REGULATION OF PYRUV

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们建议使用X射线结晶学来研究变构蛋白--兔肌丙酮酸激酶(M1-PYK)的构象变化，该蛋白在变构机制中起重要作用。尽管变构调节在使生物系统适应不断变化的环境方面很重要，但变构的分子机制却知之甚少。我们对M1-PYK变构效应的结果表明，抑制剂苯丙氨酸中的离散亚结构具有功能特定的作用，即结合所需的亚结构不同于引起变构反应的亚结构(Williams等人)。(2006)生物化学45,5421-5429)。 由此产生的假设是，在效应器结合时改变的蛋白质属性也必须划分为相同的功能子集。利用这一信息，丝氨酸和丙氨酸可以被认为是非变构效应类似物，即与苯丙氨酸竞争性结合但不引起变构反应的类似物。与苯丙氨酸相比，2-氨基丁酸有轻微的变构抑制作用。小角X射线散射数据表明，苯丙氨酸结合时观察到的整体构象变化也在非变构效应类似物结合时发生。因此，这些全局变化可能仅限于绑定功能。需要比SAXS更高的分辨率数据来表征不同效应器类似物引起的变化之间的微小差异。这些微小的差异可能是变构功能的结果。 我们以前已经解决了M1-PYK与Ala的络合物的结构在1.65？(Williams等人。(2006)生物化学45,5421-5429)。当前的波束时间请求将用于收集与苯丙氨酸、2-氨基丁酸和丝氨酸共结晶的M1-PYK的结构数据。

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. We propose to use X-ray crystallography to study conformational changes of an allosteric protein, rabbit muscle pyruvate kinase (M1-PYK) that are important to the allosteric mechanism. Despite the importance of allosteric regulation in allowing biological systems to adapt to changing environments, the molecular mechanisms of allostery are poorly understood. Our results with allosteric effectors of M1-PYK demonstrate that discrete substructures within the inhibitor, phenylalanine, have functionally specific roles, that is, substructures required for binding differ from those that elicit an allosteric response (Williams et al. (2006) Biochemistry 45, 5421-5429). The resulting hypothesis is that the protein properties that are altered upon effector binding must also be divided into the same functional subsets. Using this information, serine and alanine can be considered as non-allosteric effector analogs, analogs that bind competitively with phenylalanine but do not elicit an allosteric response. 2-amino butyric acid elicits a modest allosteric inhibition as compared to phenylalanine. Small-angle X-ray scattering data indicate that the global conformational changes observed when phenylalanine binds are also elicited when non-allosteric effector analogs bind. As such, these global changes may be confined to binding functions. Higher resolution data than are possible with SAXS are needed to characterize small differences between the changes elicited by different effector analogs. These small differences are likely consequential to allosteric functions. We have previously solved the structure of M1-PYK in complex with Ala at 1.65¿ (Williams et al. (2006) Biochemistry 45, 5421-5429). The current beamtime request will be used to collect data for the structures of M1-PYK co-crystalized with phenylalanine, 2-amino butyric acid, and serine.