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Stability and folding properties of a model β‐sheet protein, Escherichia coli CspA
Author(s) -
Reid Katherine L.,
Rodriguez Hector M.,
Hillier Brian J.,
Gregoret Lydia M.
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070228
Subject(s) - chemistry , denaturation (fissile materials) , protein folding , folding (dsp implementation) , equilibrium unfolding , crystallography , native state , phi value analysis , kinetics , urea , molten globule , circular dichroism , biochemistry , physics , electrical engineering , nuclear chemistry , engineering , quantum mechanics
Although β‐sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of β‐sheets are still not well understood. Here we examine the folding of a small, all β‐sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped‐flow fluorescence spectroscopy are monoexponential and thus also consistent with a two‐state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent‐exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.