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In this review, we will highlight technologies that enable scientists to study the molecular characteristics of tissues and/or cells without the need for antibodies or other labeling techniques. Specifically, we will focus on matrix-assisted laser desorption/ionization imaging mass spectrometry, infrared spectroscopy, and Raman spectroscopy.
Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants () for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome , which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21--progesterone, indicating that C-H bond breaking is a rate-limiting step over a 10-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
IsdGs are heme monooxygenases that break open the tetrapyrrole, releasing the iron, and thereby allowing bacteria expressing this protein to use heme as a nutritional iron source. Little is currently known about the mechanism by which IsdGs degrade heme, although the products differ from those generated by canonical heme oxygenases. A synthesis of time-resolved techniques, including in proteo mass spectrometry and conventional and stopped-flow UV/visible spectroscopy, was used in conjunction with analytical methods to define the reaction steps mediated by IsdG from Staphylococcus aureus and their time scales. An apparent meso-hydroxyheme (forming with k = 0.6 min(-1), pH 7.4, 10 mm ascorbate, 10 μm IsdG-heme, 22 °C) was identified as a likely common intermediate with the canonical heme oxygenases. Unlike heme oxygenases, this intermediate does not form with added H2O2 nor does it convert to verdoheme and CO. Rather, the next observable intermediates (k = 0.16 min(-1)) were a set of formyloxobilin isomers, similar to the mycobilin products of the IsdG homolog from Mycobacterium tuberculosis (MhuD). These converted in separate fast and slow phases to β-/δ-staphylobilin isomers and formaldehyde (CH2O). Controlled release of this unusual C1 product may support IsdG's dual role as both an oxygenase and a sensor of heme availability in S. aureus.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
It is demonstrated that time-dependent density functional theory (DFT) calculations can accurately predict changes in near-UV electronic circular dichroism (ECD) spectra of DNA as the structure is altered from the linear (free) B-DNA form to the supercoiled N-DNA form found in nucleosome core particles. At the DFT/B3LYP level of theory, the ECD signal response is reduced by a factor of 6.7 in going from the B-DNA to the N-DNA form, and it is illustrated how more than 90% of the individual base-pair dimers contribute to this strong hypochromic effect. Of the several inter-base pair parameters, an increase in twist angles is identified as to strongly contribute to a reduced ellipticity. The present work provides first evidence that first-principles calculations can elucidate changes in DNA dichroism due to the supramolecular organization of the nucleoprotein particle and associates these changes with the local structural features of nucleosomal DNA.
The conformation of an N(2)-dG adduct arising from the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent food mutagen, was determined in 5'-d(C(1)T(2)C(3)X(4)G(5)C(6)G(7)C(8)C(9)A(10)T(11)C(12))-3':5'-d(G(13)A(14)T(15)G(16)G(17)C(18)G(19)C(20)C(21)G(22)A(23)G(24))-3'; X = N(2)-dG-IQ, in which the modified nucleotide X(4) corresponds to G(1) in the 5'-d(G(1)G(2)CG(3)CC)-3' NarI restriction endonuclease site. Circular dichroism (CD) revealed blue shifts relative to the unmodified duplex, consistent with adduct-induced twisting, and a hypochromic effect for the IQ absorbance in the near UV region. NMR revealed that the N(2)-dG-IQ adduct adopted a base-displaced intercalated conformation in which the modified guanine remained in the anti conformation about the glycosidic bond, the IQ moiety intercalated into the duplex, and the complementary base C(21) was displaced into the major groove. The processing of the N(2)-dG-IQ lesion by hpol η is sequence-dependent; when placed at the reiterated G(3) position, but not at the G(1) position, this lesion exhibits a propensity for frameshift replication [Choi, J. Y., et al. (2006) J. Biol. Chem., 281, 25297-25306]. The structure of the N(2)-dG-IQ adduct at the nonreiterated G(1) position was compared to that of the same adduct placed at the G(3) position [Stavros, K. M., et al. (2014) Nucleic Acids Res., 42, 3450-3463]. CD indicted minimal spectral differences between the G(1) vs G(3) N(2)-dG-IQ adducts. NMR indicated that the N(2)-dG-IQ adduct exhibited similar base-displaced intercalated conformations at both the G(1) and G(3) positions. This result differed as compared to the corresponding C8-dG-IQ adducts placed at the same positions. The C8-dG-IQ adduct adopted a minor groove conformation when placed at position G(1) but a base-displaced intercalated conformation when placed at position G(3) in the NarI sequence. The present studies suggest that differences in lesion bypass by hpol η may be mediated by differences in the 3'-flanking sequences, perhaps modulating the ability to accommodate transient strand slippage intermediates.
Pyridoxamine (PM) is a prospective drug for the treatment of diabetic complications. In order to make zwitterionic PM more lipophilic and improve its tissue distribution, PM derivatives containing medium length alkyl groups on the hydroxymethyl side chain were prepared. The synthesis of these alkylpyridoxamines (alkyl-PMs) starting from pyridoxine offers high yields and is amenable to bulk preparations. Interestingly, alkyl-PMs were found to react with methylglyoxal (MGO), a major toxic product of glucose metabolism and autoxidation, several orders of magnitude faster than PM. This suggests the formation of nonionic pyrido-1,3-oxazine as the key step in the reaction of PM with MGO. Since the primary target of MGO in proteins is the guanidine side chain of arginine, alkyl-PMs were shown to be more effective than PM in reducing the modification of N-α-benzoylarginine by MGO. Alkyl-PMs in the presence of MGO also protected the enzymatic activity of lysozyme that contains several arginine residues next to its active site. Alkyl-PMs can be expected to trap MGO and other toxic 1,2-carbonyl compounds more effectively than PM, especially in lipophilic tissue environments, thus protecting macromolecules from functional damage. This suggests potential therapeutic uses for alkyl-PMs in diabetes and other diseases characterized by the elevated levels of toxic dicarbonyl compounds.
Amyloid fibril polymorphism is not well understood despite its potential importance for biological activity and associated toxicity. Controlling the polymorphism of mature fibrils including their morphology and supramolecular chirality by postfibrillation changes in the local environment is the subject of this study. Specifically, the effect of pH on the stability and dynamics of HET-s (218-289) prion fibrils has been determined through the use of vibrational circular dichroism (VCD), deep UV resonance Raman, and fluorescence spectroscopies. It was found that a change in solution pH causes deprotonation of Asp and Glu amino acid residues on the surface of HET-s (218-289) prion fibrils and triggers rapid transformation of one supramolecular chiral polymorph into another. This process involves changes in higher order arrangements like lateral filament and fibril association and their supramolecular chirality, while the fibril cross-β core remains intact. This work suggests a hypothetical mechanism for HET-s (218-289) prion fibril refolding and proposes that the interconversion between fibril polymorphs driven by the solution environment change is a general property of amyloid fibrils.
Lipid and lipid metabolite profiling are important parameters in understanding the pathogenesis of many diseases. Alkynylated polyunsaturated fatty acids are potentially useful probes for tracking the fate of fatty acid metabolites. The nonenzymatic and enzymatic oxidations of ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid were compared to that of linoleic and arachidonic acid. There was no detectable difference in the primary products of nonenzymatic oxidation, which comprised cis,trans-hydroxy fatty acids. Similar hydroxy fatty acid products were formed when ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid were reacted with lipoxygenase enzymes that introduce oxygen at different positions in the carbon chains. The rates of oxidation of ω-alkynylated fatty acids were reduced compared to those of the natural fatty acids. Cyclooxygenase-1 and -2 did not oxidize alkynyl linoleic but efficiently oxidized alkynyl arachidonic acid. The products were identified as alkynyl 11-hydroxy-eicosatetraenoic acid, alkynyl 11-hydroxy-8,9-epoxy-eicosatrienoic acid, and alkynyl prostaglandins. This deviation from the metabolic profile of arachidonic acid may limit the utility of alkynyl arachidonic acid in the tracking of cyclooxygenase-based lipid oxidation. The formation of alkynyl 11-hydroxy-8,9-epoxy-eicosatrienoic acid compared to alkynyl prostaglandins suggests that the ω-alkyne group causes a conformational change in the fatty acid bound to the enzyme, which reduces the efficiency of cyclization of dioxalanyl intermediates to endoperoxide intermediates. Overall, ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid appear to be metabolically competent surrogates for tracking the fate of polyunsaturated fatty acids when looking at models involving autoxidation and oxidation by lipoxygenases.
Using the 6,6'-dibromo-[1,1'-binaphthalene]-2,2'-diol molecule and its vibrational absorption (VA) and vibrational circular dichroism (VCD) spectra measured in deuterated dimethyl sulfoxide as example, we present a first detailed study of the effects induced in VCD spectra by the large-amplitude motions of solvent molecules loosely bound to a solute molecule. We show that this type of perturbation can induce significant effects in the VA and VCD spectra. We also outline a computational procedure that can effectively model the effects induced in the spectra and at the same time provide detailed structural information regarding the relative orientations of moieties involved in a solute-solvent molecular complex.
We present a systematic, multiparameter study of Rb/(129)Xe spin-exchange optical pumping (SEOP) in the regimes of high xenon pressure and photon flux using a 3D-printed, clinical-scale stopped-flow hyperpolarizer. In situ NMR detection was used to study the dynamics of (129)Xe polarization as a function of SEOP-cell operating temperature, photon flux, and xenon partial pressure to maximize (129)Xe polarization (PXe). PXe values of 95 ± 9%, 73 ± 4%, 60 ± 2%, 41 ± 1%, and 31 ± 1% at 275, 515, 1000, 1500, and 2000 Torr Xe partial pressure were achieved. These PXe polarization values were separately validated by ejecting the hyperpolarized (129)Xe gas and performing low-field MRI at 47.5 mT. It is shown that PXe in this high-pressure regime can be increased beyond already record levels with higher photon flux and better SEOP thermal management, as well as optimization of the polarization dynamics, pointing the way to further improvements in hyperpolarized (129)Xe production efficiency.