The A-state of barnase.


Abstract

The acid-induced denaturation of barnase and its mutants has been analyzed to search for partly-folded intermediates. Differential scanning calorimetry of barnase deviates from two-state behavior below pH 4.0 at low ionic strength, with the maximum discrepancy at pH 2.7. Addition of 200 mM KCl apparently restores the two-state transitions. Thermograms of barnase mutants at pH 2.7 and low ionic strength fall into three classes: alpha, symmetric transitions which fit well to a two-state equilibrium; b, asymmetric transitions indicating deviation from two-state behavior; and c, transitions with an obvious second component. The most distorted thermograms are observed for mutants that had previously been engineered to accumulate at equilibrium the major kinetic folding intermediate state of barnase at neutral pH. Further analysis of these mutants show the existence of complex equilibria on thermal denaturation. Addition of KCl leads to the slow formation of soluble aggregated forms (A-state) which share some of the properties of the "molten globule" state, i.e., significant secondary structure, lack of fixed tertiary structure, and solvent-accessible hydrophobic patches. The far-UV CD spectrum of the A-state can be explained in terms of native-like secondary structure contributions. Kinetic and chemical cross-linking experiments show that dimerization of partly-folded molecules occurs in the transition region, and such dimerization is probably the rate-limiting step in the formation of the A-state in the presence of KCl. As the A-state has been observed clearly so far for only the mutants in which the folding intermediate has been designed to accumulate, we suggest that the A-state would be related to the main folding intermediate state of barnase. The intermediate would be highly stabilized at low pH, and it is prone to self-associate in these conditions. Study holds ProTherm entries: 4798, 4799, 4800 Extra Details:

Submission Details

ID: siGaXrC73

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Sanz JM;Johnson CM;Fersht AR,Biochemistry (1994) The A-state of barnase. PMID:7727370
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