Rationally designing the accumulation of a folding intermediate of barnase by protein engineering.


A method for the stabilization of transient folding intermediates is presented. Barnase folds and unfolds via such an intermediate. Mutations that destabilize the folded state relative to the folding intermediate had been previously identified from the free energy profiles for the unfolding of mutant proteins. It is predicted that the accumulation of such mutations should lead to the intermediate being the most stable species at certain concentrations of denaturant. Mutants were prepared that contained combinations of such mutations. The behavior of these mutants on urea denaturation was studied by probes for tertiary structure (fluorescence, near-UV CD), secondary structure (far-UV CD), and hydrodynamic volume (size-exclusion chromatography). Whereas wild-type shows a two-state transition in all cases, with the same thermodynamic values being found by all probes, some of the mutants show different transitions with different structural probes. On increasing concentration of denaturant, the tertiary structure of these mutants is lost before all the secondary structure and before the protein shows the maximum expanded volume that is characteristic of the unfolded state. These mutants thus accumulate an intermediate state at equilibrium under certain urea concentrations. The intermediate state retains some degree of secondary structure but has a disrupted tertiary structure, and its degree of compactness is intermediate between the folded and the unfolded forms, probably expanding with increasing concentration of denaturant. The accumulation of the intermediate should allow its direct characterization by spectroscopy, especially NMR. Study holds ProTherm entries: 4632, 4633, 4634, 4635, 4636, 4637 Extra Details: transient folding intermediates; free energy profiles;,secondary structure; hydrodynamic volume; compactness

Submission Details

ID: GM3evb7J4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Sanz JM;Fersht AR,Biochemistry (1993) Rationally designing the accumulation of a folding intermediate of barnase by protein engineering. PMID:8257694
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