Solution NMR structure and backbone dynamics of the major cold-shock protein (CspA) from Escherichia coli: evidence for conformational dynamics in the single-stranded RNA-binding site.

The major cold-shock protein (CspA) from Escherichia coli is a single-stranded nucleic acid-binding protein that is produced in response to cold stress. We have previously reported its overall chain fold as determined by NMR spectroscopy [Newkirk, K., Feng, W., Jiang, W., Tejero, R., Emerson, S. D., Inouye, M., and Montelione, G. T. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5114-5118]. Here we describe the complete analysis of H-1, C-13, and N-15 resonance assignments for CspA, together with a refined solution NMR structure based on 699 conformational constraints and an analysis of backbone dynamics based on N-15 relaxation rate measurements. An extensive set of triple-resonance NMR experiments for obtaining the backbone and side chain resonance assignments were carried out on uniformly C-13- and N-15-enriched CspA. Using a subset of these triple-resonance experiments, the computer program AUTOASSIGN provided automatic analysis of sequence-specific backbone N, C-alpha, C', H-N, H-alpha, and side chain C-beta resonance assignments. The remaining H-1, C-13, and N-15 resonance assignments for CspA were then obtained by manual analysis of additional NMR spectra. Dihedral angle constraints and stereospecific methylene H-beta resonance assignments were determined using a new conformational grid search program, HYPER, and used together with longer-range constraints as input for three-dimensional structure calculations. The resulting solution NMR structure of CspA is a well-defined five-stranded beta-barrel with surface-exposed aromatic groups that form a single-stranded nucleic acid-binding site. Backbone dynamics of CspA have also been characterized by N-15 T-1, T-2, and heteronuclear N-15-H-1 NOE measurements and analyzed using the extended Lipari-Szabo formalism. These dynamic measurements indicate a molecular rotational correlation time tau(m), of 4.88 +/- 9.04 ns and provide evidence for fast time scale (tau(e) < 500 ps) dynamics in surface loops and motions on the microsecond to millisecond time scale within the proposed nucleic acid-binding epitope.