HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zeeb, M. Articles by Balbach, J. Search for Related Content PubMed PubMed Citation Articles by Zeeb, M. Articles by Balbach, J. Social Bookmarking                   What's this?

Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization

Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany

Reprint requests to: Jochen Balbach, Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany; e-mail: jochen.balbach{at}uni-bayreuth.de; fax: 49 921 553661.

Cold-shock proteins (CSPs) bind to single-stranded nucleic acids, thereby acting as a "RNA chaperone." To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25-mer of ssDNA (Y-Box25) using heteronuclear 2D NMR spectroscopy. Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y-Box25 interaction and were identified by extreme line broadening of their cross-peaks. Eight residues belong to the earlier proposed RNP binding motifs. A second set of seven backbone amides becomes evident by major chemical shift perturbations reporting remote conformational rearrangements upon binding. These residues are located in loop ß3–ß4 and loopß4–ß5, and include Ile18. The individual contributions of the so-identified residues were examined by fluorescence titration experiments of 15 CspB variants. Phenylalanine substitutions in- and outside the RNP motifs significantly reduce the binding affinity. Unrestricted possible backbone conformations of loop ß3–ß4 also markedly contribute to binding. Stopped-flow fluorescence kinetics revealed that the different binding affinities of CspB variants are determined by the dissociation rate, whereas the association rate remains unchanged. This might be of importance for the "RNA chaperone" activity of CspB.

Keywords: Cold-shock protein; CspB; single-stranded DNA; Y-Box; protein/ssDNA complex; NMR spectroscopy

Abbreviations: ssDNA, single-stranded DNA • HSQC, 2D ¹H/¹⁵N heteronuclear single quantum coherence spectroscopy • RNP, ribonucleoprotein • OB-fold, oligonucleotide- and/or oligosaccharide-binding fold • CspA, cold-shock protein A from Escherichia coli • CspB, cold-shock protein B from Bacillus subtilis • CSPs, cold-shock proteins • CSD, cold-shock domain • D₂O, deuterium oxide • Y-Box25, 25-mer ssDNA fragment containing the Y-Box motif ATTGG

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Sackewitz, S. von Einem, G. Hause, M. Wunderlich, F.-X. Schmid, and E. Schwarz A folded and functional protein domain in an amyloid-like fibril Protein Sci., June 1, 2008; 17(6): 1044 - 1054. [Abstract] [Full Text] [PDF]

				H. P. Morgan, P. Estibeiro, M. A. Wear, K. E.A. Max, U. Heinemann, L. Cubeddu, M. P. Gallagher, P. J. Sadler, and M. D. Walkinshaw Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach Nucleic Acids Res., May 11, 2007; 35(10): e75 - e75. [Abstract] [Full Text] [PDF]

				M. Zeeb, K. E.A. Max, U. Weininger, C. Low, H. Sticht, and J. Balbach Recognition of T-rich single-stranded DNA by the cold shock protein Bs-CspB in solution Nucleic Acids Res., September 11, 2006; 34(16): 4561 - 4571. [Abstract] [Full Text] [PDF]

				S. Katzif, E.-H. Lee, A. B. Law, Y.-L. Tzeng, and W. M. Shafer CspA Regulates Pigment Production in Staphylococcus aureus through a SigB-Dependent Mechanism J. Bacteriol., December 1, 2005; 187(23): 8181 - 8184. [Abstract] [Full Text] [PDF]