B. Ullrich (1), F. Toelgyesi (1), Z. Szeltner (2), L. Polgar (2)
and J. Fidy (1)

(1) Institute of Biophysics, Semmelweis University of Medicine, Budapest
(2) Institute of Enzymology, Biological Research Centre,
Hungarian Academy of Sciences, Budapest

We studied the effect of inhibitor-binding on the structure of HIV1
protease by its luminescence parameters such as the fluorescence
emission spectra, the quenching of fluorescence with acrylamide and
the temperature dependence of low temperature phosphorescence. The
determination of the quenching constants was realised measuring the
intensity and the lifetime of tryptophane fluorescence of the protein.

We also investigated the structural changes in the apoenzyme and
enzyme-inhibitor complex during denaturation using urea as denaturing
agent. The two tryptophanes of the native apoenzyme were found
differently accessible to the solvent and their fluorescence is
distinguishable based on the bimolecular quenching constants (kq) of
the two lifetime components. The binding of the acetyl-pepstatin
inhibitor makes the protein structure more compact and stable, the
solvent-accessible tryptophane of apoenzyme becomes more covered
during the inhibitor-binding, as shown by the decreased quenching
constant of the c1 component.

The stabilising effect of the inhibitor-binding is also confirmed by the different temperature
dependence of phosphorescence in case of the apoenzyme and the
enzyme-inhibitor complex, the freedom of motion of the tryptophane
residues is decreased in the enzyme-inhibitor complex.

This work is supported by Hungarian Grants FEFA 693, 265 and 416.



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