EXPRESSION OF FUNCTIONALITY OF ALPHA-CHYMOTRYPSIN - STRUCTURE OF A FLUORESCENT PROBE-ALPHA-CHYMOTRYPSIN COMPLEX AND THE NATURE OF ITS PH-DEPENDENCE

The 2.8-Å resolution X-ray crystallographic structure of the complex between the fluorescent probe 1-anilinonaphthalene-8-sulfonate (Ans) and α-chymotrypsin (α-CHT) has been determined at pH 3.6 and 6.6. At pH 3.6, Ans binds at a single site on the surface of the enzyme close to the amino terminus of the A chain and interacts intimately with the disulfide bond of Cys-1-122 to produce a highly fluorescent Ans-α-CHT complex. The Ans is bound to α-CHT such that it appears to be exposed to ordered solvent from one side and polar residues from the other. The fluorescence enhancement at low pH does not therefore reflect a hydrophobic site but rather one where the local dipoles do not relax substantially during the excited-state lifetime of Ans. The binding of Ans to α-CHT in crystals does not produce any major changes in the structure of the enzyme. From the similarity of the fluorescence lifetime of Ans in solutions and crystals of α-CHT and fluorescence quenching studies using PtI42- the Ans binding site has been shown to be the same in the crystal as in solution. When the pH of Ans-α-CHT crystals is raised to 6.6, the Ans and its protein environment remain essentially the same. The only structural changes occurring with change in pH are those attendant to the formation of the pH 5.4 conformer. The fluorescence decay of Ans-α-CHT crystals remains essentially the same as the pH is changed from 3.6 to 6.6. The fluorescence dependence observed with pH in solution may be due to an increase in the degree of mobility of the water molecules surrounding the Ans as the pH is increased. All of these results show that fluorescence enhancements that occur upon the binding of fluorescent probes to proteins may not always result from a hydrophobic site on the protein and suggest the need for caution in the interpretations of fluorescent changes as such probes interact with macromolecules. © 1979, American Chemical Society. All rights reserved.