DnaK, a Hsp70 homolog of Escherichia coli, together with its co-chaperones DnaJ and GrpE protects denatured proteins from aggregation and promotes their refolding by an ATP-consuming mechanism. DnaJ not only stimulates the ␥-phosphate cleavage of DnaKbound ATP but also binds polypeptide substrates on its own. Unfolded polypeptides, such as denatured luciferase, thus form ternary complexes with DnaJ and DnaK. A previous study has shown that D-peptides compete with L-peptides for the same binding site in DnaJ but do not bind to DnaK (Feifel, B., Schö nfeld, H.-J., and Christen, P. (1998) J. Biol. Chem. 273, 11999 -12002). Here we report that D-peptides efficiently inhibit the refolding of denatured luciferase by the DnaK/DnaJ/GrpE chaperone system (EC 50 ‫؍‬ 1-2 M). The inhibition of the chaperone action is due to the binding of D-peptide to DnaJ (K d ‫؍‬ 1-2 M), which seems to preclude DnaJ from forming ternary (ATP⅐DnaK) m ⅐substrate⅐DnaJ n complexes. Apparently, simultaneous binding of DnaJ and DnaK to one and the same target polypeptide is essential for effective chaperone action. The Hsp701 chaperone system of Escherichia coli includes DnaK and the two cohort proteins: DnaJ, a Hsp40 homolog, and GrpE. The chaperones assist protein folding by preventing and reversing off-pathway interactions that lead to aggregation (1). The key features of the Hsp70 chaperone system are the binding of unfolded hydrophobic segments of the target polypeptides to the ATP-liganded form of DnaK, the stabilization of the complex upon ATP hydrolysis, and the release of the bound ligands upon ADP/ATP exchange (1-3). This binding/ release cycle is controlled by DnaJ and the nucleotide exchange factor GrpE (4, 5). DnaJ interacts with DnaK through its highly conserved NH 2 -terminal J-domain and stimulates the hydrolysis of DnaK-bound ATP (2, 6). DnaJ also exerts a chaperone action on its own; upon association with denatured polypeptides, such as luciferase or rhodanese, it may prevent their aggregation (3, 6). Recently, it has been shown that D-peptides bind to DnaJ but not to DnaK (7,8). D-Peptides bind to the same site of DnaJ as L-peptides (7). Here we report that two retro-all D-peptides derived from the NH 2 -terminal segment of rhodanese inhibit the DnaK/DnaJ/GrpE chaperone system in refolding denatured firefly luciferase. EXPERIMENTAL PROCEDURESProteins-DnaK was isolated from an overproducing strain of E. coli (JM 83) bearing the plasmid pTPG9 (3). The stock solution of the protein in assay buffer (25 mM Hepes/NaOH, 100 mM KCl, pH 7.0) was stored at Ϫ80°C and contained less than 0.1 mol of ADP/mol of DnaK (9). The concentration of DnaK was determined photometrically with ⑀ 280 ϭ 14.6 mM Ϫ1 cm Ϫ1 . DnaJ and GrpE were prepared as described (10); stock solutions were stored at Ϫ80°C in 50 mM Tris/HCl, 100 mM NaCl at pH 7.7.Peptides-The peptide ala-p5 (ALLLSAPRR) was purchased with a purity of Ͼ90% from Chiron. The peptide was dissolved in 0.1% (v/v) acetic acid, 10% (v/v) acetonitrile and stored at Ϫ20°C. The two Dpeptides RI1-17 (...

Activation parameters have been determined for the intramolecular site exchange of the carbonyl groups in tricarbonyliron complexes of 1,4‐ and 2,3‐disubstituted 1,3‐butadienes, of 1,3‐cycloalkadienes and of 1,2‐dimethylidene‐cycloalkanes by temperature‐dependent 13C‐NMR. spectroscopy (see Table 1). The variations of the activation parameters are discussed in terms of a turnstile‐type mechanism for the exchange process and of the differences in the ground state and transition state stability of the complexes. A VB‐model of the complexes is used to explain the difference in the stabilities.

No abstract

On the Temperature Dependence of the 13C-NMR. Spectra of 15 -6-44 lZ, 5E)-Cyclooctadiene]tetracarbonyliron and of (lZ, 5E)-Cyclooctadiene SummaryThe activation parameters of the conformational ring inversion process (simultaneous rotation around the C (3), C (4) and C (7), C (8) bonds; cJ Scheme I) of the title compounds ( 1 and 2, respectively) have been determined between 275 K and 155 K by a complete line shape analysis of the temperature dependent proton noise-modulated decoupled I3C-NMR. spectra of 1 and 2. The temperature dependence of the rates ( k ( 1 ) and k(2), respectively) of the inversion process can be described by the following equations (no influence of the solvents was observed; Ea in J/mol): k ( l ) = 10'3,02*0,35 exp ( -(42010k 1470)/RT)sp' k ( 2 ) = 1013,05f0.17 exp ( -(33930 k 590)/RT) ') Teil der geplanten Dissertation, UniversitC de Fribourg. 58

SummaryActivation parameters have been determined for the intramolecular site exchange of the carbonyl groups in tricarbonyliron complexes of 1,4-and 2,3-disubstituted 1,3-butadienes, of 1,3-~ycloalkadienes and of 1,2-dimethylidenecycloalkanes by temperature-dependent 13C-NMR. spectroscopy (see Table 1). The variations of the activation parameters are discussed in terms of a turnstile-type mechanism for the exchange process and of the differences in the ground state and transition state stability of the complexes. A VB-model of the complexes is used to explain the difference in the stabilities.X-ray analyses of (1 -4-rp1,3-diene)tricarbonyliron complexes show the diene moiety and two carbonyl groups to occupy the basal positions in a square-based pyramidal arrangement of the ligands (cJ: e.g. [l] [2]). In solution, at room temperature, the ligands in these and related complexes undergo a rapid cyclic and concerted site exchange (LIG&~ about 38 to 53 kJmol-I) as can be seen from several recent I3C-NMR. studies (cf: [3-51 and literature cited therein). The exchange process is strongly retarded with decreasing LUMO energy of the 1,3-diene while other factors such as steric effects and the non-planarity of the diene seem to exert only minor influences. The exchange process can proceed, mechanistically, by a type of Berry pseudorotation or a type of turnstile rotation of the ligands. The two processes are indistinguishable by means of dynamic I3C-NMR. spectroscopy. The turnstile type mechanism which consists in successive 120" rotations of the tricarbonyliron fragment relative to the diene moiety, is assumed to be favoured over the Berry-type mechanism since the latter one would involve a trigonal bipyramidal intermediate with the diene ligand in one of the apical and equatorial positions -an arrangement which seems to be unrealizable for steric reasons. I ) 2,