Protein Stability, Folding, Macromolecular Associations, and Ligand Binding

Heart, Lung, And Blood Institute

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Abstract

Enzyme I: AG, EF, GP, AP[unreadable] [unreadable] 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.[unreadable] [unreadable] Currently, we are studying an important new nitrogen signal pathway of E. coli. The first enzyme of the latter pathway is enzyme I-Ntr (EI-Ntr), which after deletion of the N-terminal 127 amino acids (GAF domain), has 578 amino acids in a sequence homologous to those of other enzymes I. His356 of EI-Ntr is phosphorylated in the autocatalytic reaction of EI-Ntr with PEP/Mg(II) and this high energy phosphate is transferred to His16 of NPr, a small carrier protein containing 90 amino acids. Down stream from NPr is enzyme IIA-Ntr, which recently has been implicated in regulating the E. coli K+ channel transporter TrkA. 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. Peterkofskys laboratory. [unreadable] [unreadable] We have obtained thermodynamic parameters for the interactions of EI-Ntr (residues 170-748) and the monomeric amino terminal domain of EI-Ntr (EIN-Ntr, residues 170-424) with the acceptor protein NPr (residues 1-85) using isothermal titration calorimetry and buffers containing 10 or 50 mM Na-phosphate (pH 7.0). For EI-Ntr titrated with NPr at 293 K in the presence of 10 mM Na-phosphate, the binding stoichiometry is 1:1 and the free energy change is -7.2 +/- 0.2 kcal/mol and the enthalpy change is 1.95 kcal/mol which are approximately the same parameters observed[unreadable] for binding NPr to EIN-Ntr. When the same buffer contained 50 mM Na-phosphate at 293 K, EI-Ntr has an additional high-affinity site for binding NPr with a Gibbs free energy change of -8.9 kcal/mol and and enthalpy change of 1.6 kcal/mol. The heat capacity change for EIN-Ntr binding NPr gives an estimate of the total apolar surface area buried on complex formation that is in agreement with previous NMR estimates obtained elsewhere for the apolar surface burial that occurs on binding HPr to the sugar phosphotransferase EIN. [unreadable] [unreadable] The dimerization constant for EI-Ntr also is increased 6- to 7-fold by the presence of 50 vs. 10 mM Na-phosphate at 277 or 293 K. This is further evidence of an allosteric effect of inorganic phosphate on the conformation of EI-Ntr. However, the higher phosphate concentration only decreased the sedimentati on coefficient of the EI-Ntr dimer by 0.1 S (6.3 S; 1.5 frictional ratio). Since the dimerization constant of EI-Ntr increases 10-fold as the temperature is increased from 277 to 293 K, this range of temperature increases and decreases could be used to study the association of EI-Ntr monomers and the dissociation of the dimer, respectively. Trp fluorescence anisotropy show that the dissociation of the EI-Ntr dimer is extremely slow as the temperature is shifted from 293 to 277 K with a half-time of 15 min. As in the case of the sugar phosphotransferase enzyme I, PEP/Mg(II) binding stabilizes the dimer of EI-Ntr. [unreadable] [unreadable] DNA Binding by Cardiac-specific Nkx2.5 Homeodomain[unreadable] [unreadable] Previously, we reported on the conformational stability and energetics of DNA interactions of the cardiac-specific Csx/Nkx2.5 homeodomain (Fodor, E., Mack, J. W., Maeng, J.-S., Ju, J.-H., Lee, H. S., Gruschus, J. M., Ferretti, J. A., and Ginsburg, A. Biochemistry, 44, 12480-12490, 2005). 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. 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. [unreadable] [unreadable] 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. 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. [unreadable] [unreadable] Brain myosin Va tail fragment-DYNLL2 light chain interaction: (EF, AG, JAH, WW)[unreadable] [unreadable] 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.