Structural and dynamic aspects of beta-glycosidase from mesophilic and thermophilic bacteria by multitryptophanyl emission decay studies

The tryptophanyl emission decay of beta-glycosidase from the extremophilic archaeon Sulfolobus solfataricus (S beta gly) has been investigated by freq uency domain fluorometry. The data were analyzed in terms of sum of discret e lifetimes as well as in terms of quasi- continuous lifetime distributions of different shape. At neutral pH the emission decay is characterized by t wo components: a long-lived component, centered at 7.4 ns, and a short one at 2.7 ns, irrespective of the decay scheme used for the interpretation of the experimental results. The effects of an irreversible inhibitor, that is , cyclophellitol, and that of a powerful denaturant such as guanidinium hyd rochloride on the dynamics of S beta gly has been investigated by observing the changes induced in the two components of the tryptophanyl emission dec ay. The addition of cyclophellitol to native S beta gly reduces the contrib ution of the short-lived component but does not affect the long-lived one. Increasing concentrations of guanidinium hydrochloride differently affect t he contributions of the two emission components. Higher concentrations were required to unfold the molecular regions containing the long-lived indolic fluorophores. These results indicate that the long-lived contribution aris es from tryptophanyl residues deeply clustered in the interior of the prote in matrix, whereas the short-lived one includes residues located in less ri gid and more solvent accessible regions, some of which might be located in functionally important parts of protein. The knowledge of the crystallograp hic structure of S beta gly allowed us to evaluate some average parameters for each tryptophanyl microenvironment in the S beta gly such as hydrophobi city, structural flexibility, and ability of side chains to act as fluoresc ence quenchers. These results permitted to divide the tryptophanyl fluoresc ence of S beta gly in the contribution of two emitting groups: one consisti ng of eight closely clustered tryptophans, that is, Trp 33, 36, 60, 84, 151 174, 425, and 433, responsible for the long-lived emission component and t he other one, composed of nine tryptophans nearer to the subunit surface, t hat is, Trp 12, 156, 192, 287, 288, 316, 361, 376, 455, associable to the s hort-lived emission component. Finally, the examination of the tryptophanyl emission decay of the mesophilic beta-galactosidase from Escherichia coli (C beta gal) and the Arrhenius analysis of its dependence on temperature in dicated that the tryptophanyl environments of the mesophilic enzyme are rat her homogeneous in consequence of a larger protein dynamics. (C) 1999 Wiley -Liss, Inc.