Stability and Unfolding of Reduced Escherichia coli Glutaredoxin 2: A Monomeric Structural Homologue of the Glutathione Transferase Family † Samantha Gildenhuys, Louise A. Wallace, ‡ and Heini W. Dirr* Protein Structure-Function Research Unit, School of Molecular and Cell Biology, UniVersity of the Witwatersrand, Johannesburg 2050, South Africa ReceiVed July 7, 2008; ReVised Manuscript ReceiVed August 12, 2008 ABSTRACT: Glutaredoxin 2 (Grx2) from Escherichia coli is monomeric and an atypical glutaredoxin that takes part in the monothiol deglutathionylation of proteins. Unlike its orthologs, Grx2 is a larger molecule with a canonical glutathione transferase (GST) fold that consists of two structurally distinct domains, an N-terminal glutaredoxin domain and a C-terminal R-helical domain. While GSTs are dimeric proteins, the conformational stability and unfolding kinetics of Grx2 were investigated to establish the contribution made by the domain interface to the stability of the tertiary structure of GST-like proteins without any influence from quaternary interactions. Equilibrium unfolding transitions for Grx2, using urea as a denaturant, are monophasic and exhibit coincidence of the fluorescence and CD data indicative of a concerted loss or formation of tertiary and secondary structure. The data fit well to a two-state N T U model with no evidence that an intermediate is being formed. The experimental m value [2.7 kcal mol -1 (M urea) -1 ] is in excellent agreement with a predicted value of 2.5 kcal mol -1 (M urea) -1 based on the amount of surface area expected to become exposed during unfolding. These findings provide evidence that the two structurally distinct domains of Grx2 behave as a single cooperative folding unit. The unfolding kinetics are complex which, as a result of native-state heterogeneity, are characterized by two observable unfolding reactions that occur in parallel. A major population representing one distinct nativelike form unfolds on a fast track to denatured Grx2 with cis-Pro49. This is followed by a spectroscopically silent cis-trans proline isomerization reaction as determined by interrupted unfolding experiments. A minor population representing the other distinct nativelike form unfolds slowly with its rate being limited by an undetermined structural isomerization reaction. Further, there is no evidence indicating that unfolding proceeds via a high-energy intermediate that might suggest independent unfolding of the two nonidentical domains in Grx2. The kinetics data are, therefore, consistent with the existence of cooperativity between the domains, in agreement with the equilibrium data. The glutathione transferase (GST) 1 fold represents a large family of monomeric and dimeric intracellular proteins (http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.b.fh.b.A. html) that perform a wide range of catalytic and noncatalytic functions (for reviews, see refs 1 and 2). The canonical GST fold possesses two structural domains: a thioredoxin/glu- taredoxin-like N-terminal domain (domain 1) and a larger, unique all-R-helical C-terminal domain (domain 2) (3), as illustrated in Figure 1. The active sites of these multidomain proteins are located along the interface between the two domains with each domain contributing to overall protein function. The dynamic behavior of the domain interface also determines functional selectivity (4). Domain 1 is thought to have been recruited from a thioredoxin-like ancestral protein (3, 5), which, following the addition of all-R-helical domain 2, gave rise to a monomeric intermediate in the evolutionary pathway of the canonical dimeric GSTs (6, 7). Conformational stability data for dimeric GSTs together with their proposed evolutionary pathways suggest that the subunits of the “older” GSTs are more stable than those of the “newer” GSTs (8). As the dimer interface evolved, intersubunit interactions contribute increasingly to the stabil- ity of the individual subunits (8, 9). Although the structures and functions of the GST family have been studied exten- sively, little is known about the contribution made by the domain interface to the stability and folding of individual GST subunits. GSTs being two-domain, dimeric proteins have four components that contribute to their overall stability: the intrinsic stabilities of the two nonidentical domains, the interactions at the domain-domain interface, and the interac- tions across the dimer interface. Complicating the determi- nation of how domain interactions contribute to the stability of GST subunits is the presence of quaternary interactions † This work was supported by the University of the Witwatersrand, South African National Research Foundation Grant 205359, the South African Research Chairs Initiative of the Department of Science and Technology, and National Research Foundation Grant 64788. * To whom correspondence should be addressed. Phone: +27117176352. Fax: +27117176351. E-mail: heinrich.dirr@wits.ac.za. ‡ Current address: Genencor, A Danisco Division, 925 Page Mill Rd., Palo Alto, CA 94304. 1 Abbreviations: CD, circular dichroism; DCIP, 2,6-dichloroindophe- nol; DTT, dithiothreitol; Grx2, glutaredoxin 2; hFKBP, human FK506- binding protein; GST, glutathione transferase; k u , rate constant for unfolding; PPI, peptidyl-prolyl isomerase; ΔSASA, change in solvent accessible surface area; SCOP, Structural Classification of Proteins; SE-HPLC, size exclusion high-performance liquid chromatography; UV, ultraviolet; Xaa, any amino acid. Biochemistry 2008, 47, 10801–10808 10801 10.1021/bi801272t CCC: $40.75  2008 American Chemical Society Published on Web 09/13/2008