Fluorescence study of Escherichia coli cyclic AMP receptor protein.
Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76-83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.
Wasylewski
M
M
Department of Physical Biochemistry, Jagiellonia University, Kraków, Poland.
Małecki
J
J
Wasylewski
Z
Z
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000032:analysis*; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000494:pharmacology; D002373:Cyclic AMP Receptor Protein / Q000032:analysis*; D004926:Escherichia coli / Q000737:chemistry*; D005453:Fluorescence; D007454:Iodides / Q000494:pharmacology; D007700:Kinetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis
D000178:Acrylamides; D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D007454:Iodides; D020106:Acrylamide; D014364:Tryptophan; D000242:Cyclic AMP