Solution X-ray scattering experiments have been carried out on recombinant bovine Hsc70 (with 650 amino acid residues), a 60 kDa subfragment (residues 1-554) which has ATPase-and peptidebinding activities, a 44kDa subfragment (residues 1-386) which has only ATPase activity, and a peptidebinding fragment (residues 388-554). Modeling based on steady-state values of radii of gyration (Rg's) and P(r) functions shows that the 44 kDa and peptide-binding domains are oblate fragments while Hsc70 and

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... 60 kDa fragment are prolate and relatively elongated. R, values decreaseo significantly in the presence of MgATP relative to their values in the presence of MgADP (AR, -4-5 A) for Hsc70 and the 60 kDa fragment; in contrast, they are essentially equal in the presence of either nucleotide for the 44 kDa ATPase fragment. The kinetics of the change of R, for Hsc70 and the 60 kDa fragment under single-ATPase cycle conditions show that the transition to the ATP-induced R, occurs significantly more rapidly than ATP hydrolysis while the reverse transition to the larger R, value does not occur before product release. Altogether, the solution scattering data support a model in which a conformational change in Hsc70 (presumably to the low-peptide-affinity state) is predicated on ATP binding while the reverse transition is predicated on product release. Abstract published in Advance ACS Absrracrs, September 1, 1995. I Abbreviations: AMPPNP, 5'-adenylyl-b,y-imidodiphosphate; Hsc70, constitutively expressed stress-70 protein; MOPS, 3-(N-mopholino)propanesulfonic acid; PMSF, phenylmethanesulfonyl fluoride; R" radius of gyration; SAXS, small angle X-ray scattering; stress-70, 70 kDa heat shock-related. ATP-induced conformational change, and to attempt to correlate the conformational change with specific steps of the enzymatic ATPase cycle, we have carried out solution small-angle X-ray scattering studies of recombinant bovine Hsc70 and its subfragments. MATERIALS AND METHODS Expression and Purijication of Protein. Full-length recombinant bovine Hsc70 (amino acid residues 1 -650), which has ATPase and peptide-binding activities, and the truncation consisting of residues 1-386 ("ATPase fragment") were expressed in E. coli as described elsewhere (Wilbanks et al., 1994) . The plasmid for expressin of the truncation including residues 1-554 ("60 kDa fragment"), which also has ATPase and peptide-binding activities, was subcloned from the pT7-7-derived plasmid used to express Hsc70. This was achieved by replacing an EcoRIISaZI fragment including the codons for residues 540-650, termination signals, and part of the polylinker with a synthetic oligonucleotide encoding residues 540-554 and a termination codon. Using the same procedure, the truncation encoding residues 388-554 ("peptide-binding domain") was subcloned from a pT7-7-derived plasmid that encodes the carboxy-terminal fragment of the protein, residues 388-650 (we are indebted to Melanie 0' Brien for providing the latter plasmid). Protein expression was induced in E. coli strain BL21(DE3) by addition of isopropylthio P-D-galactoside (0.4 mM final concentration) to log phase cultures. Purification schemes for Hsc70 and the ATPase fragment were modifications of those published previously (O'Brien & McKay 1993; Wilbanks et al., 1994) . Cells were lysed by sonication, and the lysate was cleared by centrifugation at -16000g and applied to a DE-52 anion exchange column (Whatman, Clifton, NJ) equilibrated in 25 mM Tris-25 mM KC1-0.1 mM PMSF, pH 7.0. Hsc70 was eluted with 25 mM Tris-150 mM KCl-0.1 mM PMSF, pH 7.0; the ATPase fragment was eluted with a gradient of 25-200 mM KCl in the same buffer. Both proteins were next purified using an ATP-agarose column (Sigma, St. Louis, MO) as published previously, except that Hsc70 was applied to the ATP-agarose column by batch absorption with rocking for 16 h at 4 "C rather than by conventional loading of a prepoured column. Chromatofocusing over a Mono-P column followed by gel filtration on a Superdex-75 column were used as the final steps of purification (both columns from Pharmacia, Uppsala, Sweden). Final buffer conditions, established on the gel filtration column, were 10 mM MOPS-150 mM KC1, pH 7.0, for the ATPase fragment and 10 mM MOPS-100 mM K(OAc), pH 6.5, for Hsc70. A variation of this protocol was used to purify the 60 kDa fragment. Since it was not retained on the DE-52 column, it was collected in the flowthrough, applied to a hydroxylapatite column (Bio-Rad, Richmond, CA) equilibrated in 20 mM potassium phosphate-50 mM calcium phosphate-0.1 mM PMSF, pH 7, and then eluted with a gradient of 20-200 mM potassium phosphate, also with calcium and PMSF at pH 7. Chromatofocusing and gel filtration were carried out as described above; the 60 kDa protein was stored in 10 mM MOPS-150 mM KC1, pH 7.0. Cells expressing the peptide binding domain were harvested and lysed in the same fashion, and the lysate was cleared by centrifugation. The supernatant fraction from that