Stability and folding properties of a model β‐sheet protein, Escherichia coli CspA

Abstract

Although P-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of P-sheets are still not well understood. Here we examine the folding of a small, all P-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.Keywords: cold shock; fast protein folding; polyhistidine tags; protein stability; stopped-flow The challenge to understanding P-sheet structure is to decipher how residues, which may be distant from one another along the polypeptide chain, locate one another specifically and in a reasonable amount of time. To help elucidate the mechanism of sheet formation, we have characterized the folding and stability of the all P-sheet protein, CspA, the major cold shock protein of E. coli. CspA is an ideal model for /?-sheet folding studies not only because, as shown here, it is well-behaved biophysically, but also because the protein's structure represents a general single-stranded nucleic acid-binding motif. Having a firm grasp of the structurefunction relationship in this protein should aid in future protein design endeavors that could lead to the development of novel therapeutics and other molecular tools.Progress in understanding p-structure has, in part, been hampered by the difficulty in developing peptide models for sheets. Initial attempts to make isolated P-hairpins resulted in peptide aggregation because of the competition between identical intramolecular and inter-molecular interactions, and successful design of hairpins has only recently been reported

WW: An isolated three‐stranded antiparallel β‐sheet domain that unfolds and refolds reversibly; evidence for a structured hydrophobic cluster in urea and GdnHCl and a disordered thermal unfolded state

“…However, the positive peak at 230 nm, originating from the aromatic contribution to the far-UV CD spectrum~Woody, 1994; Krittanai & Johnson, 1997! as well as the negative peak above 200 nm, is consistent with the spectra exhibited by several small structurally characterized b-sheet structures, suggesting that the WW domain is folded under these conditions~the N-and C-termini are disordered in the folded structure, partially explaining the random coil-like CD spectrum!~Viguera et al, 1994; Knapp et al, 1998;Reid et al, 1998!. The thermal unfolding profile of the WW domain was monitored at 202 and 230 nm in the temperature range from 4 to 88 8C, both during the course of heat-induced unfolding and cooling-induced refolding. At 202 nm no significant changes in the magnitude of the CD signal were observed up to approximately 80 8C, but a slight loss in signal was noticed at 88 8C~Fig.…”

Is there an en route folding intermediate for cold shock proteins?

“…24 The mutation study of F20 and F31 for Csp from E. coli 13 suggested that these two residues should be present in the TS, that is, b2 and b3 might folded. This argument is also supported by the studies of Reid et al 12 that the presence of an oligonucleotide can increase the folding rate as such an oligonucleotide can bind the hydrophobic residues located in b2 and b3. Perl et al 9 have proposed that the N-terminal region is folded but the C-terminal is not in the TS.…”

“…Meanwhile, Csps have been known as a protein family that can fold fast in a two-state folding mechanism 3 as Schmid and coworkers showed that the folding dynamics of CspB from B. subtilis follows a two-state model in 1995. 4 Folding of Csps from several organisms, for example, B. subtilis, 3-8 B. caldolytics, 3,9-11 , Escherichia coli, [12][13][14][15][16] and Thermotoga maritima, 3 have been studied intensively by ensemble experiments. With the advances in single-molecule techniques, valuable insights have been gained from recent single molecule fluorescence energy transfer (FRET) studies of the Csp from T. maritima.…”

“…[17][18][19][20][21] The two-state folding dynamics has been demonstrated clearly at the single-molecule level. 17 In many experimental studies, 3,4,12 the fluorescence of a single tryptophan is used alone to probe the folding dynamics. Vu et al 15 argued that such measurements might report only on local conformational preferences and their dynamics instead of the global folding dynamics.…”

“…The dependence of rate constants of refolding and unfolding on denaturant concentration suggests that the transition state (TS) of Csp should be very compact. [3][4][5][6][7][9][10][11][12][13]15 On the other hand, f-value analysis, proposed by Fersht et.al., 23 has served as a powerful method to study the TSE. So far, f-value analysis has been used to systematically study the TSE of Csp from B. subtilis.…”