The pattern of phosphorylation of adjacent serine residues in several peptides based on the N-terminal region of human cardiac troponin I has been analysed by PAGE and 'H NMR spectroscopy to identify the products. With CAMP-dependent protein kinase, Ser24 is rapidly phosphorylated, and subsequent much slower phosphorylation of Ser23 occurs only after phosphorylation of Ser24 is almost complete. Monophosphorylation of the peptide at Ser23 was not detected at any time. On replacement of Arg22 with Ala or Met the sole phosphorylation target was Ser23, phosphorylation being considerably slower than for Ser24 in the wild-type peptide, while diphosphorylation could not be detected after prolonged incubation. The results emphasise the importance of the N-terminal sequence RRRSS for the function of cardiac troponin I and imply that in human cardiac muscle unstimulated by adrenaline, troponin I is phosphorylated on Ser24. Comparative two-dimensional NOESY data indicate that in the diphosphorylated form at physiological pH values, specific structural constraints are imposed on the N-terminal peptide region. These constraints result in the effective screening of the two phosphate groups from each other by the arginine residues N-terminal to the serine pair and stabilisation of the structure in the region of residues 25-29, which is adjacent to a site of interaction between troponin I and troponin C. These conformational changes presumably underlie the decrease in calcium sensitivity of the myofibrillar ATPase that occurs after adrenaline intervention.Keywords : troponin I ; phosphorylation ; troponin C ; NMR ; electrophoresis.Phosphorylation by the CAMP-dependent protein kinase (PKA) of adjacent serine residues in the N-terminal region of cardiac muscle troponin I (TnI) produces changes in the properties of the single Ca2+-binding site in the N-terminal domain of cardiac troponin C (TnC) with which the TnI is complexed (Robertson et al., 1982). These changes are thought to be responsible for the decrease in the Ca2' sensitivity of the myofibrillar Mg-dependent ATPase (Solar0 et al., 1976;Ray and England 1976), an effect that may play a role in the increased relaxation rate that occurs in response to P-adrenergic stimulation of intact heart. Mutation of each of the two PKA target residues (Ser22 and Ser23 in mouse, Ser23 and Ser24 in human) has indicated that both serines must be phosphorylated to obtain the characteristic decrease in CaZ+ sensitivity of force development, which can be detected in skinned cardiac muscle preparations (Zhang et al., 1995). TnI isolated from anaesthetised rabbit heart, or from unstimulated perfused heart contains approximately 1 mol phosphate/mol. With maximally stimulating concentrations of P-adrenergic agonists, the phosphate content in the perfused heart rises to approximately 2 mol/mol (Moir et al.,
Transhydrogenase couples the transfer of hydride equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The one-dimensional proton NMR spectrum of the recombinant NAD(H)-binding domain (domain I) of transhydrogenase from Rhodospirillum rubrum reveals well-defined resonances, several of which arise from a mobile loop at the protein surface. Four have been assigned to Met residues (MetA-MetD). Substitution of Met239 with either Ile (dI.M239I) or Phe (dI.M239F) resulted in loss of MetA from the NMR spectrum. Broadening and shifting of the mobile loop resonances consequent on NAD(H) binding indicate that the loop closes down on the protein surface. More NAD(H) had to be added to mutant domain I than to wild type to give comparable resonance broadening. The Kd of domain I for NADH, measured by equilibrium dialysis, was increased about three-fold by the Met239 mutations. Mutant and wild-type domain I were reconstituted with domain I-depleted membranes from R. rubrum, and with recombinant domain III of transhydrogenase. With membranes, the Km for acetylpyridine adenine dinucleotide during reverse transhydrogenation was 5x and > 6x greater in dI.M239I and dI.M239F, respectively, than in wild-type. Cyclic transhydrogenation (in membranes and the recombinant system) was substantially more inhibited (70% in dI.M239I, and 84% in dI.M239F) than either forward or reverse transhydrogenation. The docking affinities of dI.M239I and dI.M239F to the depleted membranes were similar to those of wild-type. It is concluded that Met239 is MetA in the mobile loop of domain I, and that in proteins with amino acid substitutions at this position, the binding affinity of NAD(H) is decreased, and the hydride transfer step is inhibited.
Bioenergetic properties of a mutant strain of Escherichia coli K12 designated TUV, which is resistant to the protonophoric uncoupling agent 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidiazole (TTFB) have been compared with those of its non-resistant parent, E. coli K12 Doc-S. Strain TUV grew and respired some 20-30% faster than strain Doc-S, and was cross-resistant to carbonylcyanide p-(trifluoromethoxy)phenylhydrazone and triphenyltin, but not to 2,4-dinitrophenol. Phosphorus nuclear magnetic resonance demonstrated the TTFB-mediated collapse of the transmembrane pH gradient at identical rates in starved cells of both strains, indicating that uncoupler access and function were unimpaired in the mutant under these conditions. Strain TUV displayed enhanced uncoupler resistance and maintained intracellular pH and ATP levels only when respiring. On the other hand, strain TUV also showed increased resistance to novobiocin, implying that its outer wall permeability had been lowered. We suggest that the active resistance of strain TUV results from the exclusion of uncoupler by the interaction of inner and outer membrane components in a manner modulated by the degree of cellular energization.
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