Oliveberg M, Fersht A R
Cambridge Centre for Protein Engineering, Cambridge, England, U.K.
Biochemistry. 1996 Feb 27;35(8):2738-49. doi: 10.1021/bi950967t.
New classes of small proteins have recently been found that refold rapidly with two-state kinetics from a substantially unfolded conformation ("U") and without the accumulation of a folding intermediate. Barnase, on the other hand, is representative of a class of proteins that display multistate kinetics and refold from a partly structured conformation, a folding intermediate (I). The accumulation of I on the folding pathway of barnase is highly dependent on the experimental conditions: a transition from multistate to two-state folding behavior can be induced simply by changing the reaction conditions away from physiological, i.e., elevated temperatures, high concentration of denaturant, or low pH. We argue that the change in folding behavior results from the denatured state changing under different conditions. The denatured state seems compact and partly structured at conditions that favor folding but is disorganized at denaturing conditions. At physiological pH and temperature, the denatured state (Dphys) is the folding intermediate because it is the most stable of the denatured conformation, i.e., Dphys is identical to I. At high temperature or [urea], however, Dphys becomes destabilized relative to less structured denatured states ("U"). Kinetics under these extreme conditions is two-state because the refolding reaction is from "U" to the native state with no significant accumulation of Dphys (identical to I) which is here a high-energy intermediate. The two-state behavior at low pH results from a different cause. The acid-denatured state of barnase (Dacid) is not as unfolded as "U" but energetically similar to Dphys (identical to I). It appears that protonation of Dphys has only marginal effects on its stability, so that the protonated form of Dphys constitutes the acid-denatured state at equilibrium. The energetic similarity between Dphys and Dacid gives rise to two-state kinetics at low pH, although the refolding is from a compact denatured state throughout the pH range. Protonation of Dphys to give Dacid causes the structure to become more disorganized and hydrated. The heat capacity of Dphys (identical to I) at pH 6.3 is in between that of "U" and the native protein. We suggest that protonation of folding intermediates disrupts their structural integrity and allows isoenergetic reorganizations that increase the solvation of charged residues. Such protonated and reorganized folding intermediates may then constitute the molten globules, which are compact denatured states that are sometimes observed at equilibrium at low pH and high ionic strength. Under all experimental conditions, the heat capacity of the major transition state is close to that of the native protein. This, together with its titration properties, shows that the transition state is an expanded form of the native state with a weakened but poorly hydrated hydrophobic core, and with disrupted surface regions.
最近发现了新的一类小蛋白，它们能以两态动力学从基本上未折叠的构象（“U”）迅速重新折叠，且不会积累折叠中间体。另一方面，芽孢杆菌RNA酶是一类显示多态动力学并从部分结构化构象（一种折叠中间体，I）重新折叠的蛋白质的代表。芽孢杆菌RNA酶折叠途径中I的积累高度依赖于实验条件：仅仅通过将反应条件从生理条件改变，即升高温度、增加变性剂浓度或降低pH值，就可以诱导从多态到两态折叠行为的转变。我们认为折叠行为的变化是由于变性状态在不同条件下发生了改变。在有利于折叠的条件下，变性状态似乎是紧密且部分结构化的，但在变性条件下则是无序的。在生理pH值和温度下，变性状态（Dphys）就是折叠中间体，因为它是变性构象中最稳定的，即Dphys与I相同。然而，在高温或高浓度尿素条件下，Dphys相对于结构较少的变性状态（“U”）变得不稳定。在这些极端条件下的动力学是两态的，因为重新折叠反应是从“U”到天然状态，没有Dphys（与I相同）的显著积累，这里Dphys是一个高能中间体。低pH值下的两态行为是由不同原因导致的。芽孢杆菌RNA酶的酸变性状态（Dacid）不像“U”那样完全未折叠，但在能量上与Dphys（与I相同）相似。似乎Dphys的质子化对其稳定性只有微小影响，所以Dphys的质子化形式在平衡时构成酸变性状态。Dphys和Dacid之间的能量相似性导致了低pH值下的两态动力学，尽管在整个pH范围内重新折叠都是从紧密的变性状态开始的。Dphys质子化形成Dacid会使结构变得更加无序和水合。pH值为6.3时Dphys（与I相同）的热容介于“U”和天然蛋白之间。我们认为折叠中间体的质子化破坏了它们的结构完整性，并允许等能重排，从而增加了带电残基的溶剂化。这样质子化和重新组织的折叠中间体可能构成了熔球态，熔球态是在低pH值和高离子强度下有时在平衡时观察到的紧密变性状态。在所有实验条件下，主要过渡态的热容都接近天然蛋白的热容。这一点连同其滴定性质表明，过渡态是天然状态的一种扩展形式，其疏水核心减弱但水合程度低，且表面区域被破坏。
