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A preliminary CD and NMR study of the Escherichia coli DNA polymerase III Θ subunit
Author(s) -
Li Dawei,
Allen Devon L.,
Harvey Scott,
Perrino Fred W.,
Schaaper Roel M.,
London Robert E.
Publication year - 1999
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(19990701)36:1<111::aid-prot9>3.0.co;2-1
Subject(s) - protein subunit , dna polymerase , polymerase , chemistry , microbiology and biotechnology , dna , biology , biochemistry , gene
The θ subunit of DNA polymerase III, the main replicative polymerase of Escherichia coli, has been examined by circular dichroism and by NMR spectroscopy. The polymerase core consists of three subunits: α, ϵ, and θ, with α possessing the polymerase activity, ϵ functioning as a proofreading exonuclease, and θ, a small subunit of 8.9 kD, of undetermined function. The θ subunit has been expressed in E. coli, and a CD analysis of θ indicates the presence of a significant amount of secondary structure: ∼52% alpha helix, 9% beta sheet, 21% turns, and 18% random coil. However, at higher concentrations, θ yields a poorly‐resolved 1D proton NMR spectrum in which both the amide protons and the methyl protons show poor chemical shift dispersion. Subsequent 1 H‐ 15 N HSQC analysis of uniformly‐ 15 N‐labeled θ supports the conclusion that approximately half of the protein is reasonably well‐structured. Another quarter of the protein, probably including some of the N‐terminal region, is highly mobile, exhibiting a chemical shift pattern indicative of random coil structure. The remaining amide resonances exhibit significant broadening, indicative of intermolecular and/or intramolecular exchange processes. Improved chemical shift dispersion and greater uniformity of resonance intensities in the 1 H‐ 15 N HSQC spectra resulted when [U‐ 15 N]‐θ was examined in the presence of ϵ186—the N‐terminal domain of the ϵ‐subunit. Further work is currently in progress to define the solution structure of θ and the θ–ϵ186 complex. Proteins 1999; 36:111–116. Published 1999 Wiley‐Liss, Inc.