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Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes

M. Weik^1,5, R.B.G. Ravelli², I. Silman³, J.L. Sussman⁴, P. Gros¹ and J. Kroon^1,

¹ Department of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands
² EMBL Grenoble Outstation, 38042 Grenoble Cedex 9, France
³ Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel
⁴ Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel

Reprint requests to: Dr. Martin Weik, Laboratoire de Biophysique Moléculaire, Institut de Biologie Structurale, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France; e-mail: weik{at}ibs.fr; fax: +33 4 38 78 54 94.

The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired—at least locally—sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.

Keywords: Temperature-dependent protein crystallography; dynamical transition in proteins; solvent glass transition; radiation damage; disulfide; acetylcholinesterase; enzyme radiation-inactivation

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