Role of the Chain Termini for the Folding Transition State of the Cold Shock Protein

Abstract

Residues Arg3 and Leu66 are crucially important for the enhanced stability of the cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus relative to its homologue Bs-CspB from the mesophile Bacillus subtilis. Arg3, which replaces Glu3 of Bs-CspB, accounts for two-thirds of the stability difference and for the entire difference in Coulombic interactions between the two proteins. Leu66, which replaces Glu66 of Bs-CspB, contributes additional hydrophobic interactions. To elucidate the role of these two residues near the chain termini for the rapid folding of the cold shock proteins, we performed an extensive mutational analysis of the folding kinetics to characterize interactions between residues 3, 46, and 66 in the transition state of folding. We employed a pressure-jump apparatus which allows folding to be followed over a broad range of temperatures and urea concentrations in the time range of microseconds to minutes. The N-terminal region folds early, and the interactions that originate from residue 3 are present to a large extent in the transition state already. They include a hydrophobic contribution, a general electrostatic stabilization by the positive charge of Arg3 in Bc-Csp, and a pairwise Coulombic repulsion with Glu46 in the Arg3Glu variant. The C-terminus appears to be largely unfolded in the transition state. The interactions of Leu66, including those with the already structured N-terminal region, are established only after passage through the transition state. The N- and C-termini of the cold shock proteins thus contribute differently to the folding kinetics, although they are very close in space in the folded protein.

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

“…We thus have no evidence for native state topology in collapsed unfolded CspTm, in contrast to NMR experiments on staphylococcal nuclease (8) and eglin (52) at high concentrations of urea and in contrast to suggestions from simulations (53). Similarly, there is no obvious relation to the transition state structure (54). Our observation is in contrast to recent measurements on chymotrypsin inhibitor 2 and acyl-CoA-binding protein, where indications were found for a substantial deviation of the collapsed denatured state from Gaussian chain behavior (17) possibly involving folding intermediates (55).…”

The N-terminal to C-terminal motif in protein folding and function

“…Studies of folding transition states by -analysis point to key interacting residues that have high -values (Ն0.35) in the N-and C-terminal secondary structural elements of a number of proteins. Examples include the terminal ␣-helices of acyl-coA binding protein (36), spectrin R16 (37), and the bacterial immunity proteins Im7 and Im9 (38,39); the terminal ␤-strands of muscle acylphosphatase (40), human procarboxypeptidase A2 (40,41), and PsaE (42); the terminal ␤-hairpins of protein G (43), FNIII domain 10 (44), and CspB (45,46); and the N-terminal ␣-helix and C-terminal ␤-hairpin of CI2 (47). The recently developed -analysis of folding transition states identified contacting residue pairs in the N-and C-terminal ␤-strands of ubiquitin (48,49).…”

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

“…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. Furthermore, Garcia-Mira et al 7 suggested that the development of native contacts between b1 and b4 is crucial for reaching the TS.…”

“…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.…”