Bhavesh Neel S, Sinha Ragini, Mohan P M Krishna, Hosur Ramakrishna V
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India.
J Biol Chem. 2003 May 30;278(22):19980-5. doi: 10.1074/jbc.M301615200. Epub 2003 Mar 18.
Folding studies on proteases by the conventional hydrogen exchange experiments are severely hampered because of interference from the autolytic reaction in the interpretation of the exchange data. We report here NMR identification of the hierarchy of early conformational transitions (folding propensities) in HIV-1 protease by systematic monitoring of the changes in the state of the protein as it is subjected to different degrees of denaturation by guanidine hydrochloride. Secondary chemical shifts, HN-Halpha coupling constants, 1H-15N nuclear Overhauser effects, and 15N transverse relaxation parameters have been used to report on the residual structural propensities, motional restrictions, conformational transitions, etc., and the data suggest that even under the strongest denaturing conditions (6 m guanidine) hydrophobic clusters as well as different native and non-native secondary structural elements are transiently formed. These constitute the folding nuclei, which include residues spanning the active site, the hinge region, and the dimerization domain. Interestingly, the proline residues influence the structural propensities, and the small amino acids, Gly and Ala, enhance the flexibility of the protein. On reducing the denaturing conditions, partially folded forms appear. The residues showing high folding propensities are contiguous along the sequence at many locations or are in close proximity on the native protein structure, suggesting a certain degree of local cooperativity in the conformational transitions. The dimerization domain, the flaps, and their hinges seem to exhibit the highest folding propensities. The data suggest that even the early folding events may involve many states near the surface of the folding funnel.
传统的氢交换实验用于蛋白酶的折叠研究时，由于自溶反应会干扰交换数据的解读，因此受到严重阻碍。我们在此报告，通过系统监测HIV-1蛋白酶在不同程度的盐酸胍变性条件下蛋白质状态的变化，利用核磁共振鉴定其早期构象转变（折叠倾向）的层次结构。二级化学位移、HN-Hα耦合常数、1H-15N核Overhauser效应以及15N横向弛豫参数已被用于报告残留的结构倾向、运动限制、构象转变等，数据表明即使在最强的变性条件下（6 M盐酸胍），疏水簇以及不同的天然和非天然二级结构元件也会短暂形成。这些构成了折叠核，其中包括跨越活性位点、铰链区和二聚化结构域的残基。有趣的是，脯氨酸残基会影响结构倾向，而小氨基酸甘氨酸和丙氨酸则会增强蛋白质的灵活性。在降低变性条件时，会出现部分折叠形式。显示出高折叠倾向的残基在序列上的许多位置是连续的，或者在天然蛋白质结构上彼此靠近，这表明构象转变中存在一定程度的局部协同性。二聚化结构域、侧翼及其铰链似乎表现出最高的折叠倾向。数据表明，即使是早期的折叠事件也可能涉及折叠漏斗表面附近的许多状态。
