Low resolution structure and stability studies of human GrpE#2, a mitochondrial nucleotide exchange factor Cristiano L.P. Oliveira a,b,1 , Ju ´lio C. Borges a,c,1 , Iris L. Torriani a,b , Carlos H.I. Ramos a, * a Laborato ´ rio Nacional de Luz Sı ´ncrotron, P.O. Box 6192, 13084-971 Campinas SP, Brazil b Instituto de Fı ´sica Gleb Wataghin, Universidade Estadual de Campinas, Campinas SP, Brazil c Departamento de Bioquı ´mica, Universidade Estadual de Campinas, Campinas SP, Brazil Received 14 December 2005, and in revised form 18 February 2006 Available online 9 March 2006 Abstract GrpE acts as a nucleotide exchange factor for the Hsp70 chaperone system. Only one GrpE isoform is present in Escherichia coli, but for reasons not yet well understood, two GrpE isoforms have been found in mammalian mitochondria.Therefore, studies aimed at eval- uating the physico-chemical characteristics of these proteins are important for the comprehension of the function of the Hsp70 chaperone system in different organisms. Here we report biophysical studies on human mitochondrial GrpE isoform 2. Small angle X-ray scattering measurements of human GrpE isoform 2 showed that this protein has a quaternary structure which is similar to those of human GrpE isoform 1 and E. coli GrpE: a dimer with a cruciform elongated shape. However, mitochondrial isoforms differed from each other regard- ing chemical and thermal denaturation profiles. This fact, combined with results of distinct expression patterns previously reported, point out that these proteins may have different response to external stimuli. Our results also indicate that human GrpE isoform 2 is more similar to the GrpE from E. coli than to human GrpE isoform 1. These results are relevant because differences in the conformation of Hsp70 co-chaperones are considered to be one of the reasons for functional diversity of this system. Ó 2006 Elsevier Inc. All rights reserved. Keywords: GrpE; Hsp70; Small angle X-ray scattering; Protein stability; Protein folding The Hsp70 molecular chaperone system is composed of Hsp70 and its co-chaperones Hsp40 and GrpE, which are highly expressed under situations of increased temperature and are important for several cell processes [1–4]. The Hsp70 system in Escherichia coli is known as DnaK/ DnaJ/GrpE system and has been subject of extensive in vivo and in vitro studies (see [3,4] and references therein). This system binds nascent peptide chains helping their folding, and is involved in aggregation prevention, aggregation rescue, protein translocation by membranes and degrada- tion processes, among other important functions in protein metabolism. The nucleotide binding domain (NBD) 2 of DnaK is located in its N-terminus and regulates the affinity for substrates [5,6]. The C-terminus of DnaK forms the sub- strate binding domain (SBD) and is capable of binding hydrophobic amino acid residues [6,7]. DnaJ and GrpE stimulate DnaK ATPase activity [8], and regulate the ability of DnaK to bind and stabilize unfolded proteins [9]. 0003-9861/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.02.015 * Corresponding author. Fax: +55 19 3512 1006. E-mail address: carlos.ramos@pesquisador.cnpq.br (C.H.I. Ramos). 1 These authors have contributed equally to this work. 2 Abbreviations used: Hsp, heat shock protein; EcGrpE, Escherichia coli GrpE; Mt-GrpE#1, human mitochondrial GrpE isoform 1; Mt-GrpE#2, human mitochondrial GrpE isoform 2; NBD, nucleotide binding domain of Hsp70; SBD, substrate binding domain of Hsp70; BSA, bovine serum albumin; C m , urea concentration at the midpoint of the unfolding transition; T m , temperature at the midpoint of the thermal transition; A, absorbance; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; CD, circular dichroism; [h] 222nm , residual molar ellipticity at 222 nm; AUC, analytical ultracentrifugation; SAXS, small angle X-ray scattering. www.elsevier.com/locate/yabbi ABB Archives of Biochemistry and Biophysics 449 (2006) 77–86