Thermal Measurements Of Biomolecular Systems

生物分子系统的热测量

• 批准号: 6810286
• 负责人: PHILIP D ROSS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6810286
• 关键词: DNA; bacterial virus; bioenergetics; calorimetry; capsid; heat; intermolecular interaction; ionic strengths; polynucleotides; protein protein interaction; sodium ion; stoichiometry; thermodynamics; tissue /cell culture; triple helix

In order to characterize the thermodynamics of the assembly of large macromolecular structures, we have carried out a differential scanning calorimetric study of the multiple conformational states involved in the assembly and maturation of the icosohedral head shell capsid of bacteriophage HK97. Using mutants of the gp5 head protein, we have been able to determine the energetic contributions of various structural features to the overall stability of the capsid shell. In general, for the twelve cases studied, the thermal and thermodynamic stability increases along the maturation pathway as the icosohedral capsid structure is perfected. The most outstanding result we have obtained is that the formation of covalent peptide cross-links between subunits resulting in intertwined catenated polypeptide chains produces a dramatic stabilization of the HK97 capsid structure and that the expansion transformation preceding cross-linking is energetically insignificant. This result is in marked contrast to the behavior of phages T4, T7, P22 and lambda, for which the major stabilization event is the expansion transformation. In HK97, the expansion transformation serves to facilitate the cross-link formation that greatly stabilizes the mature virus capsid (with A. C. Steven & R. L. Duda) The yeast prion protein, URE2P and its C-terminal portion (65 - 354), both in soluble and filamentous forms, all have the same thermal stability near 75degC. The difference in the enthalpy of denaturation of the complete 354 amino acid residue URE2P and the C-terminal (65 - 354) portion is approximately the energetic equivalent of two hydrogen bonds which indicates that there is only a very slight interaction of the N-terminal portion with the organized C-terminal domain. The Asp and Glu rich N- terminal domain, residues (1- 90), exhibits no cooperative melting behavior indicating that it is a disordered polypeptide chain until it is involved in amyloid formation. ( with A. Baxa & A. C. Steven) Complex formation between the transcription activator proteins TnSA and TnSC results in a 20degC increase in thermal stability. This is among the largest ever observed for protein-protein associations and is indicative of very tight binding. Thermal denaturation of the TnSA /TnSC complex is accompanied by a large positive heat capacity change that is consonant with the disruption of van der Waals interactions and the exposure to aqueous solvent of a large hydrophobic surface that is buried in the complex. These results provide thermodynamic support to the picture of the TnSA /TnSC complex obtained from the x-ray structural study that stimulated this investigation. Additionally, we have found that TnSA at physiological temperatures is partially unfolded unless complexed with TnSC. This finding calls into question certain protease assay results and their implied biological significance. ( with D.R. Ronning & F. Dyda)

为了表征大型大分子结构组装的热力学，我们已经对参与噬菌体噬菌体HK97的Icosohedral头壳壳的组装和成熟的多构象状态进行了差异扫描量热度研究。使用GP5头蛋白的突变体，我们能够确定各种结构特征对衣壳壳的整体稳定性的能量贡献。通常，在研究的十二例病例中，随着伊克西德中性衣壳结构的完善，热力学稳定性沿成熟途径增加。我们获得的最杰出结果是，在亚基之间形成了共价肽交联，导致相互交织的catenated catenated多肽链会产生HK97衣壳结构的急剧稳定，并且在交叉链接前的扩展转换在势力上是无能为力的。该结果与噬菌体T4，T7，P22和Lambda的行为形成鲜明对比，为此，主要的稳定事件是扩展变换。在HK97中，膨胀变换可促进交联的形成，从而极大地稳定了成熟的病毒capsid（使用A. C. Steven＆R。L. Duda） 酵母菌蛋白URE2P及其C末端部分（65-354），均以可溶性和丝状形式为单位，都在75DEGC附近具有相同的热稳定性。完整的354氨基酸残基URE2P和C端（65-354）部分的变性焓的差异大约是两个氢键的能量等效物，这表明N末端部分与有组织的C末端结构量只有很小的相互作用。 ASP和GLU富含N末端结构域残基（1-90）没有表现出合作的熔融行为，表明它是一个无序的多肽链，直到它参与淀粉样蛋白形成为止。 （与A. Baxa和A. C. Steven一起） 转录激活蛋白TNSA和TNSC之间的复合形成导致热稳定性增加20维。这是有史以来观察到的蛋白质蛋白缔合的最大的之一，并且表明结合非常紧密。 TNSA /TNSC复合物的热变性伴随着较大的正热容量变化，这与范德华相互作用的破坏相吻合，并暴露于大型疏水表面的水溶液中，该表面埋在该综合体中。这些结果为从X射线结构研究获得的TNSA /TNSC复合物的图像提供了热力学支持，该研究刺激了这项研究。此外，我们发现，除非与TNSC复合，否则在生理温度下的TNSA会部分展开。这一发现质疑某些蛋白酶测定结果及其隐含的生物学意义。 （与D.R. Ronning和F. Dyda一起）

项目成果

The carboxy-terminal portion of TnsC activates the Tn7 transposase through a specific interaction with TnsA.

TnsC 的羧基末端部分通过与 TnsA 的特异性相互作用激活 Tn7 转座酶。

• DOI: 10.1038/sj.emboj.7600311
• 发表时间: 2004
• 期刊: The EMBO journal
• 作者: Ronning,DonaldR;Li,Ying;Perez,ZhanitaN;Ross,PhilipD;Hickman,AlisonBurgess;Craig,NancyL;Dyda,Fred
• 通讯作者: Dyda,Fred

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA).

胸腺嘧啶的 5-甲基在聚 (dU) 和聚 (dT) 与聚 (dA) 形成的两链和三链复合物中的热力学贡献。

• DOI: 10.1002/bip.10306
• 发表时间: 2003
• 期刊: Biopolymers
• 影响因子: 2.9
• 作者: Ross,PhilipD;Howard,FrankB
• 通讯作者: Howard,FrankB