title: Free human mitochondrial GrpE is a symmetric dimer in solution.
abstract: The co-chaperone GrpE is essential for the activities of the Hsp70 system, which assists protein folding. GrpE is present in several organisms, and characterization of homologous GrpEs is important for developing structure-function relationships. Cloning, producing, and conformational studies of the recombinant human mitochondrial GrpE are reported here. Circular dichroism measurements demonstrate that the purified protein is folded. Thermal unfolding of human GrpE measured both by circular dichroism and differential scanning calorimetry differs from that of prokaryotic GrpE. Analytical ultracentrifugation data indicate that human GrpE is a dimer, and the sedimentation coefficient agrees with an elongated shape model. Small angle x-ray scattering analysis shows that the protein possesses an elongated shape in solution and demonstrates that its envelope, determined by an ab initio method, is similar to the high resolution envelope of Escherichia coli GrpE bound to DnaK obtained from single crystal x-ray diffraction. However, in these conditions, the E. coli GrpE dimer is asymmetric because the monomer that binds DnaK adopts an open conformation. It is of considerable importance for structural GrpE research to answer the question of whether the GrpE dimer is only asymmetric while bound to DnaK or also as a free dimer in solution. The low resolution structure of human GrpE presented here suggests that GrpE is a symmetric dimer when not bound to DnaK. This information is important for understanding the conformational changes GrpE undergoes on binding to DnaK.
authors.affiliation: Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Sincroton, and Departmento de Bioquímica, Instituto de Biologia, UNICAMP, Campinas, São Paulo 13084-971, Brazil.
authors.forename: Júlio C
authors.initials: JC
authors.lastname: Borges
authors.forename: Hannes
authors.initials: H
authors.lastname: Fischer
authors.forename: Aldo F
authors.initials: AF
authors.lastname: Craievich
authors.forename: Lee D
authors.initials: LD
authors.lastname: Hansen
authors.forename: Carlos H I
authors.initials: CH
authors.lastname: Ramos
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003198:Computer Simulation; D017931:DNA Primers; D019281:Dimerization; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D008928:Mitochondria / Q000737:chemistry*; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013816:Thermodynamics; D014961:X-Ray Diffraction
chemical_list: D017931:DNA Primers; C484657:GRPEL1 protein, human; C109172:GrpE-like 1 mitochondrial protein, vertebrate; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D012996:Solutions