Dumont Charles, Matsumura Yoshitaka, Kim Seung Joong, Li Jinsong, Kondrashkina Elena, Kihara Hiroshi, Gruebele Martin
Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Protein Sci. 2006 Nov;15(11):2596-604. doi: 10.1110/ps.062257406.
The lambda(6-85)() pseudo-wild type of lambda repressor fragment is a fast two-state folder (k(f) approximately 35 microsec(-1) at 58 degrees C). Previously, highly stable lambda(6-85)() mutants with k(f) > 30 microsec(-1) have been engineered to fold nearly or fully downhill. Stabilization of the native state by solvent tuning might also tune lambda(6-85)() away from two-state folding. We test this prediction by examining the folding thermodynamics and kinetics of lambda(6-85)() in a stabilizing solvent, 45% by weight aqueous ethylene glycol at -28 degrees C. Detection of kinetics by circular dichroism at 222 nm (sensitive to helix content) and small angle X-ray scattering (measuring the radius of gyration) shows that refolding from guanidine hydrochloride denatured conditions exhibits very different time scales for collapse and secondary structure formation: the two processes become decoupled. Collapse remains a low-barrier activated process, while the fastest of several secondary structure formation time scales approaches the downhill folding limit. Two-state folding of lambda(6-85)(*) is not a robust process.
λ阻遏蛋白片段的λ(6 - 85)()伪野生型是一种快速的两态折叠蛋白（在58℃时k(f)约为35微秒⁻¹）。此前，已构建出k(f) > 30微秒⁻¹的高度稳定的λ(6 - 85)()突变体，使其折叠几乎完全或完全呈下坡式。通过溶剂调节来稳定天然态可能也会使λ(6 - 85)()偏离两态折叠。我们通过研究λ(6 - 85)()在一种稳定溶剂（-28℃下45%重量比的乙二醇水溶液）中的折叠热力学和动力学来检验这一预测。通过在222 nm处的圆二色性（对螺旋含量敏感）和小角X射线散射（测量回转半径）检测动力学，结果表明从盐酸胍变性条件下复性时，折叠和二级结构形成表现出非常不同的时间尺度：这两个过程变得解耦。折叠仍然是一个低势垒的活化过程，而几个二级结构形成时间尺度中最快的接近下坡折叠极限。λ(6 - 85)(*)的两态折叠不是一个稳健的过程。
