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Reorientational Dynamics of Enzymes Adsorbed on Quartz: A Temperature-Dependent Time-Resolved TIRF Anisotropy Study

C. Czeslik^*, C. Royer, T. Hazlett and W. Mantulin

^* Universität Dortmund, Fachbereich Chemie, Physikalische Chemie I, D-44221 Dortmund, Germany; Centre de Biochimie Structurale, INSERM, CNRS, F-34090 Montpellier, France; and University of Illinois, Laboratory for Fluorescence Dynamics, Urbana, Illinois 61801 USA

Correspondence: Address reprint requests to Claus Czeslik, E-mail: claus.czeslik{at}uni-dortmund.de.

The preservation of enzyme activity and protein binding capacity upon protein adsorption at solid interfaces is important for biotechnological and medical applications. Because these properties are partly related to the protein flexibility and mobility, we have studied the internal dynamics and the whole-body reorientational rates of two enzymes, staphylococcal nuclease (SNase) and hen egg white lysozyme, over the temperature range of 20–80°C when the proteins are adsorbed at the silica/water interface and, for comparison, when they are dissolved in buffer. The data were obtained using a combination of two experimental techniques, total internal reflection fluorescence spectroscopy and time-resolved fluorescence anisotropy measurements in the frequency domain, with the protein Trp residues as intrinsic fluorescence probes. It has been found that the internal dynamics and the whole-body rotation of SNase and lysozyme are markedly reduced upon adsorption over large temperature ranges. At elevated temperatures, both protein molecules appear completely immobilized and the fractional amplitudes for the whole-body rotation, which are related to the order parameter for the local rotational freedom of the Trp residues, remain constant and do not approach zero. This behavior indicates that the angular range of the Trp reorientation within the adsorbed proteins is largely restricted even at high temperatures, in contrast to that of the dissolved proteins. The results of this study thus provide a deeper understanding of protein activity at solid surfaces.