19F-n.m.r. studies of ligand binding to 5-fluorotryptophan- and 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli

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Abstract
Two fluorine-containing analogues of the cyclic AMP receptor protein (CRP) from Escherichia coli were prepared by biosynthetic incorporation of 5-fluorotryptophan (5-F-Trp) and 3-fluorotyrosine (3-F-Tyr). The 19F-n.m.r. spectrum of the [5-F-Trp]CRP showed two signals corresponding to the two tryptophan residues, and that of the [3-F-Tyr]CRP showed six signals (two overlapping) corresponding to the six tyrosine residues; these results are as expected for a symmetrical dimer. A comparison of the 19F-n.m.r. spectra of the CRP analogues in the presence and in the absence of cyclic AMP reveals that the chemical shifts of both tryptophan residues and of two of the six tyrosine residues show differences. Since none of these residues is in direct contact with the bound nucleotide (although Trp-85 is fairly close), these shift changes must arise from induced conformational effects. The 19F-n.m.r. spectra of complexes with cyclic GMP showed chemical-shift perturbations different from those caused by cyclic AMP, indicating that different conformational changes are induced by the binding of cyclic GMP. The 19F-n.m.r. spectrum of the complex of [3-F-Tyr]CRP with tubercidin 3'',5''-(cyclic)monophosphate (which can activate transcription) showed essentially the same chemical-shift changes as seen for the cyclic AMP complex, indicating that similar conformational changes have been induced by the nucleotide binding. [3-F-Tyr]CRP in the presence of an equimolar amount of the 20 bp self-complementary DNA oligomer 5''-AATGTGAGTTAACTCACATT-3'' and excess cyclic AMP gave an 19F-n.m.r. spectrum that was almost identical with that for the [3''F-Tyr)CRP-cyclic AMP complex, indicating that the binding of DNA does not induce significant conformational changes involving the tyrosine residues. Proteolysis of [3-F-Tyr]CRP with chymotrypsin produced a 31 kDa fragment that is a dimer containing the cyclic AMP-binding domain. This fragment contains five of the six tyrosine residues, and its 19F-n.m.r. chemical shifts were essentially the same as those of the intact protein except for one missing signal (signal F): this signal could be assigned to Tyr-206 and shown to be unpertubed by the binding of cyclic nucleotide to the intact [3-F-Tyr]CRP. The similarity of the 19F-n.m.r. chemical shifts in the .alpha.-fragment and the intact CRP indicates that the .alpha.-fragment retains the same structure as found in the intact protein. The binding of cyclic AMP to the .alpha.-fragment caused chemical-shift perturbations of signals from the same two residues (TyrA and TyrB) that are influenced in complexes with intact CRP, with similar but not identical perturbations being observed. The overall results provide evidence for specific conformational changes in protein when different cyclic nucleotides bind to CRP, but give no indication of further conformational changes in the .alpha.-core of the protein when DNA binding takes place.

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