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N Juranic

Publications and source records attributed to N Juranic.

6 recordsLinked to original sources
PubMed2001

Cellular energetics in the preconditioned state: protective role for phosphotransfer reactions captured by 18O-assisted 31P NMR.

Cell survival is critically dependent on the preservation of cellular bioenergetics. However, the metabolic mechanisms that confer resistance to injury are poorly understood. Phosphotransfer reactions integrate ATP-consuming with ATP-producing processes and could thereby contribute to the generation of a protective phenotype. Here, we used ischemic preconditioning to induce a stress-tolerant state and (18)O-assisted (31)P nuclear magnetic resonance spectroscopy to capture intracellular phosphotransfer dynamics. Preconditioning of isolated perfused hearts triggered a redistribution in phosphotransfer flux with significant increase in creatine kinase and glycolytic rates. High energy phosphoryl fluxes through creatine kinase, adenylate kinase, and glycolysis in preconditioned hearts correlated tightly with post-ischemic functional recovery. This was associated with enhanced metabolite exchange between subcellular compartments, manifested by augmented transfer of inorganic phosphate from cellular ATPases to mitochondrial ATP synthase. Preconditioning-induced energetic remodeling protected cellular ATP synthesis and ATP consumption, improving contractile performance following ischemia-reperfusion insult. Thus, the plasticity of phosphotransfer networks contributes to the effective functioning of the cellular energetic system, providing a mechanism for increased tolerance toward injury.

Adenosine Triphosphate↗
PubMed2000

Compromised energetics in the adenylate kinase AK1 gene knockout heart under metabolic stress.

Rapid exchange of high energy carrying molecules between intracellular compartments is essential in sustaining cellular energetic homeostasis. Adenylate kinase (AK)-catalyzed transfer of adenine nucleotide beta- and gamma-phosphoryls has been implicated in intracellular energy communication and nucleotide metabolism. To demonstrate the significance of this reaction in cardiac energetics, phosphotransfer dynamics were determined by [(18)O]phosphoryl oxygen analysis using( 31)P NMR and mass spectrometry. In hearts with a null mutation of the AK1 gene, which encodes the major AK isoform, total AK activity and beta-phosphoryl transfer was reduced by 94% and 36%, respectively. This was associated with up-regulation of phosphoryl flux through remaining minor AK isoforms and the glycolytic phosphotransfer enzyme, 3-phosphoglycerate kinase. In the absence of metabolic stress, deletion of AK1 did not translate into gross abnormalities in nucleotide levels, gamma-ATP turnover rate or creatine kinase-catalyzed phosphotransfer. However, under hypoxia AK1-deficient hearts, compared with the wild type, had a blunted AK-catalyzed phosphotransfer response, lowered intracellular ATP levels, increased P(i)/ATP ratio, and suppressed generation of adenosine. Thus, although lack of AK1 phosphotransfer can be compensated in the absence of metabolic challenge, under hypoxia AK1-knockout hearts display compromised energetics and impaired cardioprotective signaling. This study, therefore, provides first direct evidence that AK1 is essential in maintaining myocardial energetic homeostasis, in particular under metabolic stress.

Adenosine Triphosphate↗
PubMed2000

Structure-fluorescence correlations in a single tryptophan mutant of carp parvalbumin: solution structure, backbone and side-chain dynamics.

Heterogeneous fluorescence intensity decays of tryptophan in proteins are often rationalized using a model which proposes that different rotameric states of the indole alanyl side-chain are responsible for the observed fluorescence lifetime heterogeneity. We present here the study of a mutant of carp parvalbumin bearing a single tryptophan residue at position 102 (F102W) whose fluorescence intensity decay is heterogeneous and assess the applicability of a rotamer model to describe the fluorescence decay data. We have determined the solution structure of F102W in the calcium ligated state using multi-dimensional nuclear magnetic resonance (NMR) and have used the minimum perturbation mapping technique to explore the possible existence of multiple conformations of the indole moiety of Trp102 of F102W and, for comparison, Trp48 of holo-azurin. The maps for parvalbumin suggest two potential conformations of the indole side-chain. The high energy barrier for rotational isomerization between these conformers implies that interwell rotation would occur on time-scales of milliseconds or greater and suggests a rotamer basis for the heterogeneous fluorescence. However, the absence of alternate Trp102 conformers in the NMR data (to within 3 % of the dominant species) suggests that the heterogeneous fluorescence of Trp102 may arise from mechanisms independent of rotameric states of the Trp side-chain. The map for holo-azurin has only one conformation, and suggests a rotamer model may not be required to explain its heterogeneous fluorescence intensity decay. The backbone and Trp102 side-chain dynamics at 30 degrees C of F102W has been characterized based on an analysis of (15)N NMR relaxation data which we have interpreted using the Lipari-Szabo formalism. High order parameter (S(2)) values were obtained for both the helical and loop regions. Additionally, the S(2) values imply that the calcium binding CD and EF loops are not strictly equivalent. The S(2) value for the indole side-chain of Trp102 obtained from the fluorescence, NMR relaxation and minimum perturbation data are consistent with a Trp moiety whose motion is restricted.

Amino Acid Substitution↗
PubMed2000

Quantitative determination of magnetization exchange rate constants from a series of two-dimensional exchange NMR spectra

A method for quantitative determination of magnetization exchange rate constants (cross-relaxation and chemical exchange) from a series of two-dimensional exchange spectra is presented. The method, the least error matrix analysis (LEMA), combines a series of full matrix calculations at different mixing times in a least-squares manner. LEMA embodies the principal advantages of full-relaxation matrix analysis (FMA) and initial rate buildup (BU) analysis. Like FMA, it takes into account all the relations among the spectral matrix elements and in analogy to BU makes use of their time evolution. By means of calculations, simulations, and experiments, we have shown that LEMA provides the dynamic matrix from a given set of experimental data with errors that are smaller than in either FMA or BU calculations.

Journal Article↗
PubMedDate not supplied

Simulated dipeptide recognition by vancomycin.

The antimicrobial activity of vancomycin and related glycopeptide antibiotics is due to stereospecific recognition of polypeptide components in bacterial cell walls. To better understand how these antibiotics recognize polypeptide determinants, we have developed dynamic models of the complexes formed by the vancomycin aglycon and two different dipeptide ligands, Ac-D-ala-D-ala and Ac-D-ala-gly. Molecular dynamics simulations of the two complexes, initially conditioned with distance constraints derived from two-dimensional nuclear magnetic resonance (NMR) studies, are conformationally stable and propagate in a manner consistent with the NMR-derived constraints after the constraints are removed. Free energy calculations accurately predict the relative binding affinity of these two complexes and help validate the simulation models for detailed structural analysis. Although the two ligands adopt similar conformations when bound to the antibiotic, there are clear differences in the configuration of intermolecular hydrogen bonds, the overall shape of the antibiotic, and other structural features of the two complexes. This analysis illustrates how complex structural and dynamic factors interrelate and contribute to differences in binding affinity.

Anti-Bacterial Agents↗