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Methionine Adenosyltransferase -Helix Structure Unfolds at Lower Temperatures than ß-Sheet: A 2D-IR Study

Ibon Iloro ^*, Rosana Chehín ^*, Félix M. Goñi ^*, María A. Pajares  and José-Luis R. Arrondo ^*

^* Unidad de Biofísica (Centro Mixto CSIC-UPV) and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain; and Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Madrid, Spain

Correspondence: Address reprint requests to José-Luis R. Arrondo, Tel.: 34-946-012-485; Fax: 34-944-648-500; E-mail: gbproarj{at}lg.ehu.es.

Two-dimensional infrared spectroscopy has been used to characterize rat liver methionine adenosyltransferase and the events taking place during its thermal unfolding. Secondary structure data have been obtained for the native recombinant enzyme by fitting the amide I band of infrared spectra. Thermal denaturation studies allow the identification of events associated with individual secondary-structure elements during temperature-induced unfolding. They are correlated to the changes observed in enzyme activity and intrinsic fluorescence. In all cases, thermal denaturation proved to be an irreversible process, with a T_m of 47–51°C. Thermal profiles and two-dimensional infrared spectroscopy show that unfolding starts with -helical segments and turns, located in the outer part of the protein, whereas extended structure, associated with subunit contacts, unfolds at higher temperatures. The data indicate a good correlation between the denaturation profiles obtained from activity measurements, fluorescence spectroscopy, and the behavior of the infrared bands. A study of the sequence of events that takes place is discussed in light of the previous knowledge on methionine adenosyltransferase structure and oligomerization pathway.