Abstract:
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Oliveberg M,Fersht ARdoi
10.1021/bi950967tsubject
Has Abstractpub_date
1996-02-27 00:00:00pages
2738-49issue
8eissn
0006-2960issn
1520-4995pii
bi950967tjournal_volume
35pub_type
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