Bhatia Sandhya, Krishnamoorthy G, Udgaonkar Jayant B
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru 560065, India.
Phys Chem Chem Phys. 2018 Jan 31;20(5):3216-3232. doi: 10.1039/c7cp06214a.
Protein folding/unfolding transitions between the native (N) and unfolded (U) states are usually describable as two-state, only because of the dominant presence of the N and/or U states, because of which high energy intermediates remain undetected. Delineation of the cooperativity underlying these transitions, and characterization of high energy intermediates that are populated sparsely, have been difficult challenges, especially under equilibrium conditions, and require the use of a sensitive probe that reports on both the structures and population distributions of the partially unfolded intermediates. In this study, the use of multisite time-resolved FRET to monitor structural change in five specific segments of the small protein monellin, has brought out local deviations from two-state behavior during unfolding. It is shown that in some segments of the protein structure, denaturant-induced unfolding proceeds first by gradual expansion of the N state, then by an all-or-none transition from the N state ensemble to the U state ensemble, followed finally by expansion of the U state. Segments encompassing the sole helix appear, however, to unfold completely through a gradual transition from the N to U states. Finally, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the β-sheet.
蛋白质在天然态（N）和未折叠态（U）之间的折叠/去折叠转变通常被描述为两态过程，这仅仅是因为N态和/或U态占主导地位，使得高能量中间体未被检测到。描绘这些转变背后的协同性以及表征稀少出现的高能量中间体一直是艰巨的挑战，尤其是在平衡条件下，这需要使用一种能够报告部分未折叠中间体的结构和种群分布的灵敏探针。在本研究中，使用多位点时间分辨荧光共振能量转移（FRET）来监测小蛋白质莫内林五个特定片段的结构变化，揭示了去折叠过程中偏离两态行为的局部偏差。结果表明，在蛋白质结构的某些片段中，变性剂诱导的去折叠首先通过N态的逐渐扩展进行，然后通过从N态集合到U态集合的全或无转变，最后是U态的扩展。然而，包含唯一螺旋的片段似乎是通过从N态到U态的逐渐转变而完全去折叠的。最后，研究表明莫内林的平衡去折叠不仅是异质的，而且唯一的α螺旋和β折叠的不同部分之间的非协同程度也不同。
