CRP subunit association and hinge conformation changes in response to cAMP binding: analysis of C-helix cysteine-substituted CRP.
We investigated the characteristics of 13 CRP variants having cysteine substituted at positions 113, 115, 116, 117, 118, 120, 122, 124, 126, 127, 129, 130, or 131, positions that span the length of the CRP C alpha-helix. Under reducing conditions, the WT and all Cys-substituted forms of CRP migrated as 23.5 kDa CRP monomer species on SDS-PAGE gels. In the absence of a reductant, 9 of 13 Cys-substituted forms of CRP including the L113C, S117C, M120C, L124C, V126C, T127C, E129C, K130C, and V131C CRP contained protein that migrated as 47 kDa CRP dimer species on SDS-PAGE gels. CNBr digestion of the protein preparations followed by MALDI-TOF MS analysis of the peptide fragments showed these 47 kDa species to be CRP dimers that originated from disulfide bonds formed between positional-pair C alpha-helix Cys residues. The ratio of monomer CRP and disulfide cross-linked CRP within a Cys-substituted CRP preparation was found to be independent of cAMP for Cys-substituted CRP preparations denatured and renatured in the presence of various cAMP concentrations. This finding suggests that there is no large-scale concerted motion (i.e., scissoring) of the CRP subunits in response to cAMP binding. In addition, we have identified three amino acid residues located along the CRP C alpha-helix that play a role in facilitating the conformation transition of the CRP hinge from that characteristic of apo-CRP to that characteristic of the CRP.cAMP complex.
Tomlinson
Steven R
SR
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409, USA.
Tutar
Yusuf
Y
Harman
James G
JG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein; D003545:Cysteine / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D014157:Transcription Factors; C481662:crp protein, E coli; D000242:Cyclic AMP; D003545:Cysteine