Biomedical subjects

Mariana N Dimitrova

Publications and source records attributed to Mariana N Dimitrova.

5 recordsLinked to original sources

PubMed2005

Human mitochondrial ClpP is a stable heptamer that assembles into a tetradecamer in the presence of ClpX.

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 (s(20,w) 7.0 S; M(app) 169, 200) at concentrations > or = 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 equilibrium sedimentation measurements has a M(app) of 1 x 10(6). 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.

Adenosine Triphosphate↗

PubMed2004

Oxidation of methionine residues in the prion protein by hydrogen peroxide.

Reaction of H(2)O(2) with the recombinant SHa(29-231) prion protein resulted in rapid oxidation of multiple methionine residues. Susceptibility to oxidation of individual residues, assessed by mass spectrometry after digestion with CNBr and lysC, was in general a function of solvent exposure. Met 109 and Met 112, situated in the highly flexible amino terminus, and key residues of the toxic peptide PrP (106-126), showed the greatest susceptibility. Met 129, a residue located in a polymorphic position in human PrP and modulating risk of prion disease, was also easily oxidized, as was Met 134. The structural effect of H(2)O(2)-induced methionine oxidation on PrP was studied by CD spectroscopy. As opposed to copper catalyzed oxidation, which results in extensive aggregation of PrP, this reaction led only to a modest increase in beta-sheet structure. The high number of solvent exposed methionine residues in PrP suggests their possible role as protective endogenous antioxidants.

Animals↗

PubMed2003

Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli.

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(2+)-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate-binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg(2+)) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 degrees C, 5 mM pyruvate (with 2 mM Mg(2+)) has the opposite effect. 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, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg(2+) is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 10(8) to < 5 x 10(5) or 3 x 10(7) M(-1), respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg(2+) also increases domain coupling and the dimerization constant approximately 42-fold. With 2 mM Mg(2+) at 15-25 degrees C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K(A)' approximately 10(6) M(-1) (deltaG' = -8.05 +/- 0.05 kcal/mole and deltaH = +3.9 kcal/mole at 20 degrees C); deltaC(p) = -0.33 kcal K(-1) mole(-1). The binding of PEP to EI(H189A) is synergistic with that of Mg(2+). Thus, physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-enzyme I.

Binding Sites↗

PubMed2003

Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin.

A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.

Amino Acid Sequence↗

PubMed2002

Interdomain interaction and substrate coupling effects on dimerization and conformational stability of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5 and 4 and 20 degrees C. Concurrently, thermal unfolding of these forms of EI has been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [ approximately 2 x 10(6) (M monomer)(-1)] compared to those of dephospho-EI and EI(H189A) at 20 degrees C. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl(2) [K(A)' > or = 10(8) M(-1) at 4 or 20 degrees C], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg(2+) also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A), increasing the transition temperature (T(m)) for unfolding the C-terminal domain by approximately 18 degrees C and that for the N-terminal domain by approximately 9 degrees C to T(max) congruent with 63 degrees C, giving a value of K(D)' congruent with 3 microM PEP at 45 degrees C. PEP alone also promotes the dimerization of EI(H189A) but only increases T(m) approximately 5 degrees C for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated value of K(D)' congruent with 0.2 mM for PEP dissociation in the absence of Mg(2+) at 45 degrees C. In contrast, the dimerization constant of phospho-EI at 20 degrees C is the same in the absence and presence of 5 mM PEP and 2 mM MgCl(2). Thus, the separation of substrate binding effects from those of phosphorylation by studies with the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg(2+) are important determinants of both the conformational stability and dimerization of dephospho-EI.

Amino Acid Substitution↗