Backbone dynamics of TFE-induced native-like fold of region 4 of Escherichia coli RNA polymerase sigma70 subunit.
Folding of a recombinant protein rECsigma(70) (4) comprising domain 4 of E. coli RNA polymerase sigma(70) subunit, upon addition of 2,2,2-trifluoroethanol (TFE) to its aqueous solution, was monitored by heteronuclear NMR spectroscopy. The TFE-induced migration of resonance signals in a series of (15)N-HSQC spectra displayed sequence-dependent heterogeneity. A common trend of uniform upfield shift in both (1)H and (15)N dimensions, indicative of generation of helical structures, breaks down for some residues almost cooperatively at 10-15% TFE (v/v), pointing to the buildup of non-helical regions separating the initially induced helices. The preferences of residues to assume either helical or non-helical conformation are correlated with the location in the sequence rather than with their type. CSI descriptors and (15)N relaxation data obtained for the protein at 10% TFE allowed characterization of the stability of the pre-folded state of rECsigma(70) (4). By all the criteria applied, three highly populated alpha-helical regions separated by much more flexible residues forming a loop and a turn in the DNA-binding HLHTH motif were identified. The location of the secondary structure elements along the protein sequence coincides with those found in homologous proteins, and with the helix nucleation regions determined in unfolded rECsigma(70) (4) at low pH. The bimodal distribution of the (15)N relaxation parameters enabled identification of residues forming a framework of the folded protein strictly corresponding to the HLHTH motif, bracketed by unfolded terminal regions. Thus, in respect to rECsigma(70) (4) in aqueous solution TFE acts not only as a strong helix inducer, but also as a folding agent.
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
Piotr
P
Kaczka
Agnieszka
A
Polkowska-Nowakowska
Krystyna
K
Bolewska
Igor
I
Zhukov
Jarosław
J
Poznański
Kazimierz L
KL
Wierzchowski
D000595:Amino Acid Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D016415:Sequence Alignment; D012808:Sigma Factor / Q000737:chemistry* / Q000378:metabolism; D014270:Trifluoroethanol / Q000737:chemistry*
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D012808:Sigma Factor; D014270:Trifluoroethanol; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases