Investigating Protein Hydration and Structure with Azide Probes

产品说明：这项研究的长期目标是使用振动探针和红外光谱来研究蛋白质的水合作用和结构。所提出的叠氮化物是灵敏的、位点特异性的和非微扰的振动探针，具有足够的空间和时间分辨率来研究感兴趣的分子动力学。叠氮化物探针将用于研究三种蛋白质中的水合动力学：腺苷酸激酶（AK）、超折叠绿色荧光蛋白（sfGFP）和磷酸丙糖异构酶（TIM）。AK突变与溶血性贫血、网状细胞发育不全和纤毛运动障碍有关。TIM缺乏导致慢性溶血性贫血、心肌病、感染易感性、神经功能障碍和人类寿命缩短，来自寄生虫布氏锥虫的TIM已被鉴定为昏睡病的病原体。 本论文的研究内容包括腺苷叠氮化物1a-1f和苯丙氨酸叠氮化物2a-2 e的合成。叠氮化物1作为AK中水合和静电的有效IR探针的能力将被研究 通过线性IR，然后发送到合作者，马修塔克，二维IR分析，以测量周围的叠氮化物探针水合动力学。叠氮化物2具有刚性接头，其允许叠氮化物和残基侧链之间的距离以受控和系统的方式变化。叠氮化物2将被合成并遗传地并入sfGFP和TIM中，其中它们将充当分子水合统治者。线性红外光谱将用于研究这些蛋白质的水合和静电，合作者Christopher Cheatum将进行二维红外实验，以测量叠氮化物探针周围的水合动力学。 叠氮化物的生物正交反应性将用于通过与炔金属羰基的点击反应在sfGFP中的150位点处连接叠氮化物2b，以直接比较叠氮化物和金属羰基作为振动水合探针。 将使用与AK结合的叠氮化物1和与AK结合的叠氮化物2，用2D IR光谱研究从它们之间的非谐耦合强度提取两个叠氮化物同位素异构体之间的距离和角度的能力。合作者Steven Corcelli将使用计算方法来模拟水合动力学，并将帮助开发一个模型来描述叠氮基之间非谐耦合的距离依赖性。 这项健康相关研究的另一个目标是培养至少六名本科生的合成，生物化学和光谱技术。

DESCRIPTION: The long-term objective of this research is to use vibrational probes and infrared (IR) spectroscopy to study protein hydration and structure. The proposed azides are sensitive, site- specific, and non-perturbative vibrational probes with sufficient spatial and temporal resolution to study the molecular dynamics of interest. The azide probes will be used to investigate hydration dynamics in three proteins: adenylate kinase (AK), superfolder green fluorescent protein (sfGFP), and triosephosphate isomerase (TIM). Mutations in AK are associated with hemolytic anemia, reticular dysgenesis, and ciliary dyskinesia. TIM deficiency results in chronic hemolytic anaemia, cardiomyopathy, susceptibility to infections, neurological dysfunction, and decreased life spans in humans and TIM from the parasite Trypanosoma brucei has been identified as the causative agent of sleeping sickness. The proposed research involves the synthesis of adenosine azides 1a-1f and phenylalanine azides 2a-2e. The ability of azides 1 to serve as effective IR probes of hydration and electrostatics in AK will be investigated by linear IR and then sent to a collaborator, Matthew Tucker, for 2D IR analysis to measure hydration dynamics around the azide probe. Azides 2 have rigid linkers that allow the distance between the azide and the residue side chain to be varied in a controlled and systematic fashion. Azides 2 will be synthesized and genetically incorporated into sfGFP and TIM where they will serve as molecular hydration rulers. Linear IR spectroscopy will be used to investigate the hydration and electrostatics of these proteins and a collaborator, Christopher Cheatum, will perform 2D IR experiments to measure hydration dynamics around the azide probe. The bioorthogonal reactivity of azides will be used to ligate azide 2b at the 150 site in sfGFP by a click reaction with an alkyne metal carbonyl to directly compare azides and metal carbonyls as vibrational hydration probes. The ability to extract the distance and angle between two azide isotopomers from the strength of anharmonic coupling between them will be investigated with 2D IR spectroscopy using azides 1 bound to AK and azides 2 incorporated into AK. Collaborator Steven Corcelli will use computational methods to model hydration dynamics and will aid in the development of a model to describe the distance dependence of anaharmonic coupling between azido groups. Another goal of this health related research is to train a minimum of six undergraduate students in synthetic, biochemical, and spectroscopic techniques.