19F n.m.r. studies of conformational changes accompanying cyclic AMP binding to 3-fluorophenylalanine-containing cyclic AMP receptor protein from Escherichia coli.
A fluorine-containing analogue of the cyclic AMP (cAMP) receptor protein (CRP) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorophenylalanine (3-F-Phe). 19F n.m.r. studies on this protein have provided direct evidence for cAMP-induced conformational changes not only within the cAMP-binding domain but also within the hinge region connecting the cAMP-binding domain to the DNA-binding headpiece. At 313 K, the 19F n.m.r. spectrum of [3-F-Phe]CRP showed five signals corresponding to the five phenylalanine residues as expected for a symmetrical dimer. Proteolysis of [3-F-Phe]CRP with subtilisin produced a fragment (the alpha-fragment) containing the cAMP-binding domain. The alpha-fragment contains all the phenylalanines except for Phe-136, a residue located in the hinge region. By comparing the 19F spectra of [3-F-Phe]CRP and its alpha-fragment, the signal for Phe-136 was assigned. The chemical shifts of the corresponding signals in the two spectra are similar, indicating that the alpha-fragment retains the structure it has in the intact protein. The largest cAMP-induced shift was observed for the signal from Phe-136 providing direct evidence for a conformational change in the hinge region. However, whereas binding of a single cAMP molecule to a CRP dimer is known to be sufficient to activate the DNA binding, the n.m.r. data indicate that the hinge region does not have the same conformation in both subunits when only one cAMP molecule is bound.
Hinds
M G
MG
Laboratory of Molecular Structure, National Institute for Medical Research, Mill Hill, London, U.K.
King
R W
RW
Feeney
J
J
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005461:Fluorine; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008968:Molecular Conformation; D010446:Peptide Fragments / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D011953:Receptors, Cyclic AMP / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011953:Receptors, Cyclic AMP; D005461:Fluorine; C027218:3-fluorophenylalanine; D010649:Phenylalanine; D000242:Cyclic AMP