Browsing by Subject "NMR spectroscopy"

Now showing 1 - 4 of 4
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    Direct affinity of dopamine to lipid membranes investigated by Nuclear Magnetic Resonance spectroscopy
    (Elsevier, 2016-04) Matam, Yashasvi; Ray, Bruce D.; Petrache, Horia I.; Department of Physics, School of Science
    Dopamine, a naturally occurring neurotransmitter, plays an important role in the brain’s reward system and acts on sensory receptors in the brain. Neurotransmitters are contained in lipid membraned vesicles and are released by exocytosis. All neurotransmitters interact with transport and receptor proteins in glial cells, on neuronal dendrites, and at the axonal button, and also must interact with membrane lipids. However, the extent of direct interaction between lipid membranes in the absence of receptors and transport proteins has not been extensively investigated. In this report, we use UV and NMR spectroscopy to determine the affinity and the orientation of dopamine interacting with lipid vesicles made of either phosphatidylcholine (PC) or phosphatidylserine (PS) lipids which are primary lipid components of synaptic vesicles. We quantify the interaction of dopamine's aromatic ring with lipid membranes using our newly developed method that involves reference spectra in hydrophobic environments. Our measurements show that dopamine interacts with lipid membranes primarily through the aromatic side opposite to the hydroxyl groups, with this aromatic side penetrating deeper into the hydrophobic region of the membrane. Since dopamine's activity involves its release into extracellular space, we have used our method to also investigate dopamine's release from lipid vesicles. We find that dopamine trapped inside PC and PS vesicles is released into the external solution despite its affinity to membranes. This result suggests that dopamine's interaction with lipid membranes is complex and involves both binding as well as permeation through lipid bilayers, a combination that could be an effective trigger for apoptosis of dopamine-generating cells.
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    Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity
    (MDPI, 2016-12) Andrews, Forest H.; Wechsler, Cindy; Rogers, Megan P.; Meyer, Danilo; Tittmann, Kai; McLeish, Michael J.; Department of Chemistry and Chemical Biology, School of Science
    Benzoylformate decarboxylase (BFDC) and pyruvate decarboxylase (PDC) are thiamin diphosphate-dependent enzymes that share some structural and mechanistic similarities. Both enzymes catalyze the nonoxidative decarboxylation of 2-keto acids, yet differ considerably in their substrate specificity. In particular, the BFDC from P. putida exhibits very limited activity with pyruvate, whereas the PDCs from S. cerevisiae or from Z. mobilis show virtually no activity with benzoylformate (phenylglyoxylate). Previously, saturation mutagenesis was used to generate the BFDC T377L/A460Y variant, which exhibited a greater than 10,000-fold increase in pyruvate/benzoylformate substrate utilization ratio compared to that of wtBFDC. Much of this change could be attributed to an improvement in the Km value for pyruvate and, concomitantly, a decrease in the kcat value for benzoylformate. However, the steady-state data did not provide any details about changes in individual catalytic steps. To gain insight into the changes in conversion rates of pyruvate and benzoylformate to acetaldehyde and benzaldehyde, respectively, by the BFDC T377L/A460Y variant, reaction intermediates of both substrates were analyzed by NMR and microscopic rate constants for the elementary catalytic steps were calculated. Herein we also report the high resolution X-ray structure of the BFDC T377L/A460Y variant, which provides context for the observed changes in substrate specificity.
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    Peptide Model of the Mutant Proinsulin Syndrome. II. Nascent Structure and Biological Implications
    (Frontiers Media, 2022-03-01) Yang, Yanwu; Glidden, Michael D.; Dhayalan, Balamurugan; Zaykov, Alexander N.; Chen, Yen-Shan; Wickramasinghe, Nalinda P.; DiMarchi, Richard D.; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    Toxic misfolding of proinsulin variants in β-cells defines a monogenic diabetes syndrome, designated mutant INS-gene induced diabetes of the young (MIDY). In our first study (previous article in this issue), we described a one-disulfide peptide model of a proinsulin folding intermediate and its use to study such variants. The mutations (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe 1H and 1H-13C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic 13C-labeling. Parent spectra retain nativelike features in the neighborhood of the single disulfide bridge (cystine B19-A20), including secondary NMR chemical shifts and nonlocal nuclear Overhauser effects. This partial fold engages wild-type side chains LeuB15, LeuA16 and PheB24 at the nexus of nativelike α-helices α1 and α3 (as defined in native proinsulin) and flanking β-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > SerB24 >> ProA16 > ProB15 (least organized). The same order pertains to (a) overall α-helix content as probed by circular dichroism, (b) synthetic yields of corresponding three-disulfide insulin analogs, and (c) ER stress induced in cell culture by corresponding mutant proinsulins. These findings suggest that this and related peptide models will provide a general platform for classification of MIDY mutations based on molecular mechanisms by which nascent disulfide pairing is impaired. We propose that the syndrome's variable phenotypic spectrum-onsets ranging from the neonatal period to later in childhood or adolescence-reflects structural features of respective folding intermediates.
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    Rates and Intermediates in Ser26 Mutants of Benzoylformate Decarboxylase
    (Office of the Vice Chancellor for Research, 2016-04-08) Lian, NiLen T.; McLeish, Michael J.; Ray, Bruce D.
    Benzoylformate decarboxylase (BFDC), a thiamine diphosphate dependent enzyme, catalyzes decarboxylation of benzoylformate to benzaldehyde and CO2. The BFDC reaction proceeds through at least four individual chemical steps and, recently, NMR spectroscopy has been used to measure the ratios of intermediates in the overall reaction. This method permits calculation of rate constants for formation of the first intermediate, mandelylThDP (k2) and its subsequent decarboxylation (k3), as well as the combined breakdown of the enamine and product release (k4). As part of a study of the contributions of the active site residues, Ser26, His70 and His281, to the individual catalytic steps several Ser26 variants were expressed and purified. Initially, the variants were characterized using steady-state kinetics. Subsequently, the enzymes were mixed with benzoylformate and the mixture immediately acid quenched to trap intermediates of the reaction. NMR spectroscopy was used to identify and quantitate individual catalytic intermediates. Rate constants for the formation of these intermediates were then determined and compared to those of the wild-type enzyme. Here we report those results and discuss their implications for the role of Ser26 in the BFDC reaction mechanism.