Sequence-specific proton NMR assignment and secondary structure of the Arc repressor of bacteriophage P22, as determined by two-dimensional proton NMR spectroscopy

Abstract

The Arc repressor of bacteriophage P22 is a DNA binding protein that does not belong to any of the known classes of such proteins. We have undertaken a 1H NMR study of the protein with the aim of elucidating its three-dimensional structure in solution and its mode of binding of operator DNA. Here we present the 1H nuclear magnetic resonance (NMR) assignments of all backbone protons and most of the side-chain protons of Arc repressor. Elements of secondary structure have been identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser enhancements (NOEs). Two alpha-helical regions have been found in the peptide regions 16-29 and 35-45. The ends of the helices could not yet be firmly established and could extend to residue 31 for the first helix and to residue 49 for the second. Immediately before the first helix, between residues 8 and 14, a region is present with beta-sheet characteristics dominated by a close proximity of the alpha-protons of residues 9 and 13. Because of the dimeric nature of the protein there are still two possible ways in which the NOEs in the beta-sheet region can be interpreted. If the NOEs are intramonomer, this requires a tight turn involving residues 10-12. Alternatively, if the NOEs are intermonomer, then and antiparallel beta-sheet would be implicated comprising two strands of different Arc monomers. While the data presently do not allow an unambiguous choice between these two possibilities, some evidence is discussed that favors the latter (beta-sheet between monomers).(ABSTRACT TRUNCATED AT 250 WORDS)

Molten-globule conformation of Arc repressor monomers determined by high-pressure 1H NMR spectroscopy.

“…The starting point for our analysis is the refined three-dimensional structure of the Arc repressor dimer with its intermonomer 1-sheet as described by R. Kaptein (personal communication). Our results indicate a predissociation conformational change and suggest that the molten globule ofArc repressor monomer adopts the structure containing a (3-strand, -y-turn, (-strand features (15,16) in the 8-to 14-residue region that is believed to be the DNA-binding site.…”

“…NMR has been shown to be a powerful tool for the investigation ofprotein structure and dynamics of protein folding intermediates (14). The complete 1H NMR assignments for Arc repressor have been reported (15,16) and a tertiary and quaternary structure model for Arc repressor has been proposed (12).…”

Rational Design, Synthesis, and DNA Binding Properties of Novel Sequence‐Selective Peptidyl Congeners of Ametantrone

“…There exist however precedents for proteins that bind to DNA in the major groove and adopt a b-sheet conformation, as in the case of the dimeric Arc repressor. [27,28] The biological evaluation of the new compounds showed that the presence of the lysine-containing peptide in the AMderived series, although necessary for sequence-specific DNA recognition, confers a decisively high hydrophilic character, impairing biological effects in the cell lines tested. However, the residual cytotoxicity exhibited by the highly charged compound 15 points to the possibility of exploiting appropriate delivery systems to circumvent cell permeation.…”

1H NMR assignments of apo calcyclin and comparative structural analysis with calbindin D9k and S100β

“…Only those NOEs that could be unambiguously assigned as strictly intermolecular were used for the subsequent calculations of the dimer structure. We note that other homodimeric protein and peptide systems, for which only homonuclear 'H NMR data were available, have been analyzed in a similar manner (e.g., Breg et al, 1989;Kay et al, 1991 ;Shaw et al, 1992).…”