title: Frequency-domain fluorescence studies of an extracellular metalloproteinase of Staphylococcus aureus.
abstract: A frequency-domain fluorescence study of calcium-binding metalloproteinase from Staphylococcus aureus has shown that this two-tryptophan-containing protein exhibits a double-exponential fluorescence decay. At 10 degrees C in 0.05 M Tris-HCl buffer (pH 9.0) containing 10 mM CaCl2, fluorescence lifetimes of 1.2 and 5.1 ns are observed. Steady-state and frequency-domain solute-quenching studies are consistent with the assignment of the two lifetimes to the two tryptophan residues. The tryptophan residue characterized by a shorter lifetime has a maximum of fluorescence emission at about 317 nm and the second one exhibits a maximum of its emission at 350 nm. These two residues contribute almost equally to the protein's fluorescence. These results, as well as fluorescence-quenching studies using KI and acrylamide as a quencher, indicate that in calcium-loaded metalloproteinase, the tryptophan residue characterized by the shorter lifetime is extensively buried within the protein. The second residue is exposed on the surface of the protein. The tryptophan residues of metalloproteinase have acrylamide dynamic-quenching rate constants, kq values, of 2.3 and 0.26 X 10(9) M-1 X s-1 for the exposed and buried residue, respectively. A study of the temperature dependence of the fluorescence lifetime for the two tryptophan components gives activation energies, Ea values, for thermal quenching of 1.8 and 2.2 kcal/mol for the buried and the exposed residue, respectively. Dissociation of Ca2+ from the protein causes a change in the protein's structure, as can be judged from dramatic changes which occur in the fluorescence properties of the buried tryptophan residue. These changes include an approx. 13 nm red-shift in the maximum of the fluorescence emission and an increase in the acrylamide-quenching rate constant, and they indicate that the removal of Ca2+ results in an increase in the exposure and the polarity of the microenvironment of this 'blue' residue.
mesh_terms: D002118:Calcium / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D005453:Fluorescence; D008666:Metalloendopeptidases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000201:enzymology*; D013816:Thermodynamics; D014364:Tryptophan; D015032:Zinc / Q000378:metabolism
chemical_list: D005029:Ethylenediamines; D014364:Tryptophan; D004492:Edetic Acid; D008666:Metalloendopeptidases; D015032:Zinc; D002118:Calcium; C034269:tetraethylenepentamine