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Articles by Boris G. Dzikovski in JoVE
JoVE Bioengineering
पता लगाने और गैस चरण मुख्यधारा सिगरेट के धुएँ में फ्री रेडिकल्स की सफाई के लिए एक प्रोटोकॉल
1CDCF-AOX Lab, 2National Biomedical Center for Advanced ESR Technology (ACERT), Department of Chemistry and Chemical Biology, Cornell University
स्पिन - फँसाने ESR स्पेक्ट्रोस्कोपी संयंत्र एंटीऑक्सिडेंट लाइकोपीन, Pycnogenol और गैस चरण सिगरेट के धुएं में मुक्त कण की सफाई पर अंगूर बीज निकालने के प्रभाव का अध्ययन करने के लिए इस्तेमाल किया गया था.
Other articles by Boris G. Dzikovski on PubMed
Oxygen Permeation Profile in Lipid Membranes: Comparison with Transmembrane Polarity Profile
Permeation of oxygen into membranes is relevant not only to physiological function, but also to depth determinations in membranes by site-directed spin labeling. Spin-lattice (T(1)) relaxation enhancements by air or molecular oxygen were determined for phosphatidylcholines spin labeled at positions (n = 4-14, 16) of the sn-2 chain in fluid membranes of dimyristoyl phosphatidylcholine, by using nonlinear continuous-wave electron paramagnetic resonance (EPR). Both progressive saturation and out-of-phase continuous-wave EPR measurements yield similar oxygen permeation profiles. With pure oxygen, the T(2)-relaxation enhancements determined from homogeneous linewidths of the linear EPR spectra are equal to the T(1)-relaxation enhancements determined by nonlinear EPR. This confirms that both relaxation enhancements occur by Heisenberg exchange, which requires direct contact between oxygen and spin label. Oxygen concentrates in the hydrophobic interior of phospholipid bilayer membranes with a sigmoidal permeation profile that is the inverse of the polarity profile established earlier for these spin-labeled lipids. The shape of the oxygen permeation profile in fluid lipid membranes is controlled partly by the penetration of water, via the transmembrane polarity profile. At the protein interface of the KcsA ion channel, the oxygen profile is more diffuse than that in fluid lipid bilayers.
Spin-labeled Gramicidin A: Channel Formation and Dissociation
Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35 degrees C (the gel to Pbeta phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 A, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the Pbeta and Lalpha phases of DPPC (above 35 degrees C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (approximately 0.4-1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.
Oxygen Profiles in Membranes
Transmembrane profiles of molecular oxygen in lipid bilayers are not only significant for membrane physiology and pathology, but also are essential to the determination of membrane protein structure by site-directed spin labeling. Oxygen profiles obtained with spin-labeled lipid chains have a Boltzmann sigmoidal dependence on the depth into each lipid leaflet, which represents a two-compartment distribution between outer and inner regions of the membrane, with a transfer free energy that depends linearly on distance from the dividing planes. Transmembrane profiles for intramembrane polarity, and for water penetration into the membrane, have an identical form, but are of the reverse sign. Comparison with recently published oxygen profiles from a site-specifically spin-labeled alpha-helical transmembrane peptide validates the use of spin-labeled lipids for all these profiles and provides the necessary bridge to generate the full bilayer from a single lipid leaflet.
Channel and Nonchannel Forms of Spin-labeled Gramicidin in Membranes and Their Equilibria
Channel and nonchannel forms of gramicidin A (GA) were studied by ESR in various lipid environments using new mono- and double-spin-labeled compounds. For GA channels, we demonstrate here how pulse dipolar ESR can be used to determine the orientation of the membrane-traversing molecule relative to the membrane normal and to study subtle effects of lipid environment on the interspin distance in the spin-labeled gramicidin channel. To study nonchannel forms of gramicidin, pulse dipolar ESR was used first to determine interspin distances corresponding to monomers and double-helical dimers of spin-labeled GA molecules in the organic solvents trifluoroethanol and octanol. The same distances were then observed in membranes. Since detection of nonchannel forms in the membrane is complicated by aggregation, we suppressed any dipolar spectra from intermolecular interspin distances arising from the aggregates by using double-labeled GA in a mixture with excess unlabeled GA. In hydrophobic mismatching lipids (L(β) phase of DPPC), gramicidin channels dissociate into free monomers. The backbone structure of the monomeric form is similar to a monomeric unit of the channel dimer. In addition to channels and monomers, the double-helical conformation of gramicidin is present in some membrane environments. In the gel phase of saturated phosphatidylcholines, the fraction of double helices increases in the following order: DLPC < DMPC < DSPC < DPPC. The equilibrium DHD/monomer ratio in DPPC was determined. In membranes, the double-helical form is present only in aggregates. In addition, we studied the effect of N-terminal substitution in the GA molecule upon channel formation. This work demonstrates how pulsed dipolar ESR may be utilized to study complex equilibria of peptides in membranes.
Conformational Distributions and Hydrogen Bonding in Gel and Frozen Lipid Bilayers: A High Frequency Spin-Label ESR Study
The ESR parameters of PC spin labels in frozen membranes do not simply represent the membrane polarity or water penetration profile. Instead, they show a distribution between hydrogen-bonded (HB) and non-hydrogen bonded (non-HB) states, which is affected by a number of factors in the membrane composition. Similar to the exclusion of solutes from crystallizing solvents, the pure bulk gel phase excludes nitroxides, forcing acyl chains to take bent conformations. In these conformations the nitroxide is hydrogen-bonded. Furthermore, upon gradual cooling in the supercooled gel PC labels undergo slow lateral aggregation resulting in a broad background signal. However, if the sample is instantly frozen, this background is replaced by the HB component. In membranes with cholesterol the observed HB/ non-HB ratio can best be described by a partition-like equilibrium between nitroxides located in defects of lipid structure within the hydrophobic core and those close to the membrane surface.
