Protein Stability, Folding, Macromolecular Associations,

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Enzyme I: [AG, EF, GP, AP] The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(II)-dependent autophosphorylation by PEP requires the homodimer. Previously, we showed that PEP and Mg(II) binding strongly promotes dimer formation whereas pyruvate and Mg(II) binding favored the monomer. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, was observed. Currently, we are studying an important new nitrogen signal pathway of E. coli, which involves phosphorylation of a His residue in the first enzyme (EI-Ntr, 66.7 kDa) by PEP in the presence of Mg(II) and a subsequent transfer of the high-energy phosphate to a small histidine-containing NPr protein (9.1 kDa) . These studies are in collaboration with G. Wang (University of Nebraska Medical Center) who is determining NMR structures and Alan Peterkofsky (NHLBI) who is expressing proteins cloned by Yeong-Jae Seok (Dir., Microbiology, Seoul National University), who is working during summer months in A. Peterkofsky?s laboratory. We are conducting biophysical and thermodynamic measurements on the purified EI-Ntr (which has been expressed without the N-terminal GAF domain) and on the binding of NPr to EI-Ntr (using isothermal titration calorimetry). DNA Binding by Cardiac-specific Nkx2.5 Homeodomain Previously, we reported on the conformational stability and energetics of DNA interactions of the cardiac-specific Csx/Nkx2.5 homeodomain (EF, AG, JMG, JWM, J-SM, J-HJ, HSL, JAF). The amino acid sequence of Nkx2.5 is 73% identical with the parent vnd/NK-2 homeodomain protein from Drosophila melanogaster previously studied in our laboratory by Gonzalez et al. (Biochemistry 40, 4923-4931, 2001). Both proteins specifically bind to 5'-CAAGTG-3' in duplex DNA. The NMR structures for DNA complexes of Nkx2.5(C56S) and NK-2 are the same although unbound NK-2 has been found to be more flexible than free Nkx2.5(C56S). DNA binding by Nkx2.5(C56S) is enthalpically controlled. Titrations of specific 18 bp duplex DNA with the cardiac-specific homeodomain Nkx2.5(C56S) have utilized an ultrasensitive isothermal titration calorimeter (ITC). During the latter studies, we found that impaired DNA or unfolded protein can be detected by ITC (EF, AG). Namely, as the free DNA neared depletion, we observed large apparent decreases in the binding enthalpy when the DNA was impaired or when the temperature was sufficiently high to produce some unfolding of the free protein. Either effect can be attributed to refolding of the biopolymer that occurs as a result of stabilization due to the large favorable change in free energy on HD binding to DNA (-11.8 kcal/mol at 298 K). In either case, thermodynamic parameters obtained in such ITC experiments are unreliable. By using a lower temperature (85 vs. 95 C) during the annealing of complementary DNA strands, damage of the 18 bp duplex DNA (transition T = 72 C) is avoided, and titrations with the homeodomain are normal at temperatures from 10 to 40 C when >95 % of the protein is folded. Under the latter conditions, the heat capacity change (-0.19 kcal/deg.mol) is linear with increasing temperature and is more negative than that calculated from the burial of solvent accessible surface areas (-0.15 ? 0.01 kcal/deg.mol), consistent with water structures being at the protein-DNA interfaces. The work of Dr. Fodor is particularly important because mutations in the human NKX2.5 homeodomain (identical in sequence to the mouse Nkx2.5 HD) have been found to produce abnormalities in human embryonic heart development and, in the future, the specific defects of such mutants will be probed. Human mitochondrial ClpP protease and ClpX chaperone interaction: (MND, AG, MRM, SGK) The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (7.0 S; 169,200 Mr) at concentrations >3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by sedimentation equilibrium measurements has an apparent molecular weight of 1,000,000. Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles. Brain myosin Va tail fragment-DYNLL2 light chain interaction: (EF, AG, JAH, WW) The myosin Va light chain DYNLL2 has been proposed to function as an adaptor to link myosin to certain cargo. When Dr. Hammer approached us to help characterize the interaction of a brain myosin V tail fragment (myoVa, 28.6 kDa dimer) with DYNLL2 (20.8 kDa dimer), we used far-UV circular dichroism (CD) and analytical ultracentrifugal techniques. CD measurements revealed a DYNLL2-induced change in the secondary structure of this dimeric myosin fragment containing the Exon B motif that is consistent with a-helical coiled-coil formation. The binding of DYNLL2 also was found to increase the thermal stability of this myosin Va fragment. Analytical ultracentrifugation yielded an apparent association constant of ~3,000,000 /M for dimeric DYNLL2 binding to the dimeric myosin Va fragment. Thus, the binding of DYNLL2 to the DDK sequence of Exon B can produce local structural changes in the tail region of brain myosin Va.