Biomedical subjects

Janez Strancar

Publications and source records attributed to Janez Strancar.

11 recordsLinked to original sources

PubMed2006

Motional restrictions of membrane proteins: a site-directed spin labeling study.

Site-directed mutagenesis was used to produce 27 single cysteine mutants of bacteriophage M13 major coat protein spanning the whole primary sequence of the protein. Single-cysteine mutants were labeled with nitroxide spin labels and incorporated into phospholipid bilayers with increasing acyl chain length. The SDSL is combined with ESR and CD spectroscopy. CD spectroscopy provided information about the overall protein conformation in different mismatching lipids. The spin label ESR spectra were analyzed in terms of a new spectral simulation approach based on hybrid evolutionary optimization and solution condensation. This method gives the residue-level free rotational space (i.e., the effective space within which the spin label can wobble) and the diffusion constant of the spin label attached to the protein. The results suggest that the coat protein has a large structural flexibility, which facilitates a stable protein-to-membrane association in lipid bilayers with various degrees of hydrophobic mismatch.

Amino Acid Substitution↗

PubMed2005

Effects of lipid composition on the membrane activity and lipid phase behaviour of Vibrio sp. DSM14379 cells grown at various NaCl concentrations.

The membrane lipid composition of living cells generally adjusts to the prevailing environmental and physiological conditions. In this study, membrane activity and lipid composition of the Gram-negative bacterium Vibrio sp. DSM14379, grown aerobically in a peptone-yeast extract medium supplemented with 0.5, 1.76, 3, 5 or 10% (w/v) NaCl, was determined. The ability of the membrane to reduce a spin label was studied by EPR spectroscopy under different salt concentrations in cell suspensions labeled with TEMPON. For lipid composition studies, cells were harvested in a late exponential phase and lipids were extracted with chloroform-methanol-water, 1:2:0.8 (v/v). The lipid polar head group and acyl chain compositions were determined by thin-layer and gas-liquid chromatographies. (31)P-NMR spectroscopy was used to study the phase behaviour of the cell lipid extracts with 20 wt.% water contents in a temperature range from -10 to 50 degrees C. The results indicate that the ability of the membrane to reduce the spin label was highest at optimal salt concentrations. The composition of both polar head groups and acyl chains changed markedly with increasing salinity. The fractions of 16:0, 16:1 and 18:0 acyl chains increased while the fraction of 18:1 acyl chains decreased with increasing salinity. The phosphatidylethanolamine fraction correlated inversely with the lysophosphatidylethanolamine fraction, with phosphatidylethanolamine exhibiting a minimum, and lysophosphatidylethanolamine a maximum, at the optimum growth rate. The fraction of lysophosphatidylethanolamine was surprisingly high in the lipid extracts. This lipid can form normal micellar and hexagonal phases and it was found that all lipid extracts form a mixture of lamellar and normal isotropic liquid crystalline phases. This is an anomalous behaviour since the nonlamellar phases formed by total lipid extracts are generally of the reversed type.

Biophysical Phenomena↗

PubMed2004

Phospholipid mesophases at solid interfaces: in-situ X-ray diffraction and spin-label studies.

In this work, we report on recent investigations, both on the global and on the local molecular architecture of supported phospholipid model membranes. A brief theoretical introduction explains how global structural information on supramolecular lipid ensembles can be retrieved from surface X-ray diffraction measurements as well as how spin-label electron paramagnetic resonance spectroscopy (EPR) provides complementary information on the local environment of probe molecules. The combination of especially designed X-ray cells with the technique of small- and wide-angle X-ray surface scattering makes it possible to explore various fields of lipid research and its applications. Examples for different physico-chemical conditions are presented: (i) in situ chemistry under excess of water conditions demonstrating how solid-supported lipid films sense salinity, (ii) the 3D electron density reconstruction of a vesicle-fusion intermediate under controlled humidity, and (iii) complementary temperature and pressure effects on oriented phospholipid samples. Further, special attention has been given to the influence of different film preparation techniques with respect to quality and the defect structure manifestation. To resolve the proportions and local properties of defects in a hydrated lipid-deposited surface, spin-label EPR was applied. The results from 9.6 GHz EPR as well as from 1.2 GHz EPR suggest the alignment to be in the range between 30% and 80%. In addition, slow time-dependent EPR measurements point to nano-structural rearrangements due to water flow and reduction of alignment quality.

Cell Membrane↗

PubMed2004

The membrane lateral domain approach in the studies of lipid-protein interaction of GPI-anchored bovine erythrocyte acetylcholinesterase.

A novel membrane lateral domain approach was used to test whether the activity of the membrane-bound enzyme acetylcholinesterase (AChE) depends on the local properties (e.g. local lipid ordering) of bovine erythrocyte-ghost membrane. This issue has an additional aspect of interest due to an alternative mode of insertion of AChE molecules into the membrane by the glycosylphosphatidylinositol (GPI) anchor. In our experiments the lateral domain membrane structure was influenced by temperature and by the addition of n-butanol, and was quantitatively characterized using the method of EPR spectrum decomposition. The activity of AChE was determined by a colorimetric assay in the same samples. The results show that the membrane stabilizes the conformation of the membrane-bound AChE compared to the isolated AChE. In addition, a correlation was observed between the temperature dependence of order parameter of the most-ordered domain type and the activity of AChE. Therefore, our findings support the idea that the function of GPI proteins can be modulated by the lipid bilayer. Based on the assumption that the overall activity of AChE depends on the order parameters of particular domain types as well as their proportions, two models for AChE activity were introduced. In the first, a random distribution of enzyme molecules was proposed, and in the second, localization of enzyme molecules in a single (cholesterol-rich) domain type was assumed. Better agreement between measured and calculated activity values speaks in favor of the second model.

Acetylcholinesterase↗

PubMed2003

Quantification of binding of some thiol-reactive clenbuterol analogues to bovine serum albumin by electron paramagnetic resonance spectroscopy.

The only free thiol group of bovine serum albumin (BSA) was coupled in a high yield with some novel thiol-reactive clenbuterol analogues. The unreacted cysteines were probed with maleimide spin label to determine the yield of the coupling reaction. A novel approach to determining free thiol groups of BSA quantitatively by electron paramagnetic resonance spectroscopy and spectral decomposition without the usual gel-filtration step or extensive dialysis is presented.

Animals↗

PubMed2002

Quantifying the lateral lipid domain properties in erythrocyte ghost membranes using EPR-spectra decomposition.

Using EPR spectroscopy a typical lateral domain structure was detected in the membranes of spin-labeled bovine erythrocyte ghosts. The spectral parameters were determined by decomposing the EPR spectrum into three spectral components and tuned by a hybrid-evolutionary-optimization method. In our experiments the lateral domain structure and its properties were influenced by the variation in the temperature and by the addition of n-butanol. The specific responses of the particular domain types were detected. For the most-ordered domain type a break was seen in the temperature dependence of its order parameter, while the order parameters of the two less-ordered domain types exhibited a continuous decrease. Below the break-point temperature the alcohol-induced membrane fluidity variation is mainly a consequence of the change in the proportions of the least- and the most-ordered domain type and not the change of the domain-type ordering or dynamics (with n-butanol concentration). On the other hand, the fluidity variation above the break-point temperature arises from both types of changes. Interestingly, the proportion of the domain type that has its order parameter between that of the least- and the most-ordered domain type remains almost constant with concentration as well as with temperature, which implies its stability. Such characterization of the lateral membrane domain structure could be beneficial when considering the lipid-protein interactions, because it can be assumed that the activity of the membrane-bound enzyme depends on the properties of the particular domain type.

Animals↗

PubMed2002

Electron paramagnetic resonance characterization of the exopolysaccharide layer produced by bacteria.

In order to establish a method to characterize the structural heterogeneity of the bacterial surface, research was conducted with a combination of experiments based on electron paramagnetic resonance (EPR) concentration-imaging (CI) and the modeling of translational diffusion with local restrictions. The benefits and drawbacks of this approach are discussed for the Vibrio sp. exopolysaccharide (EPS) layer.

Cell Wall↗

PubMedDate not supplied

Spin label EPR-based characterization of biosystem complexity.

Following the widely spread EPR spin-label applications for biosystem characterization, a novel approach is proposed for EPR-based characterization of biosystem complexity. Hereto a computational method based on a hybrid evolutionary optimization (HEO) is introduced. The enormous volume of information obtained from multiple HEO runs is reduced with a novel so-called GHOST condensation method for automatic detection of the degree of system complexity through the construction of two-dimensional solution distributions. The GHOST method shows the ability of automatic quantitative characterization of groups of solutions, e.g. the determination of average spectral parameters and group contributions. The application of the GHOST condensation algorithm is demonstrated on four synthetic examples of different complexity and applied to two physiologically relevant examples--the determination of domains in biomembranes (lateral heterogeneity) and the study of the low-resolution structure of membrane proteins.

Algorithms↗

PubMedDate not supplied

Exploring the local conformational space of a membrane protein by site-directed spin labeling.

Molecular modeling based on a hybrid evolutionary optimization and an information condensation algorithm, called GHOST, of spin label ESR spectra was applied to study the structure and dynamics of membrane proteins. The new method is capable of providing detailed molecular information about the conformational space of the spin-labeled segment of the protein in a membrane system. The method is applied to spin-labeled bacteriophage M13 major coat protein, which is used as a model membrane protein. Single cysteine mutants of the coat protein were labeled with nitroxide spin labels and incorporated in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers. The new computational method allows us to monitor distributions of local spatial constraints and molecular mobility, in addition to information about the location of the protein in a membrane. Furthermore, the results suggest that different local conformations may coexist in the membrane protein. The knowledge of different local conformations may help us to better understand the function-structure relationship of membrane proteins.

Algorithms↗

PubMedDate not supplied

Speeding up a genetic algorithm for EPR-based spin label characterization of biosystem complexity.

Complexity of biological systems is one of the toughest problems for any experimental technique. Complex biochemical composition and a variety of biophysical interactions governing the evolution of a state of a biological system imply that the experimental response of the system would be superimposed of many different responses. To obtain a reliable characterization of such a system based on spin-label Electron Paramagnetic Resonance (EPR) spectroscopy, multiple Hybrid Evolutionary Optimization (HEO) combined with spectral simulation can be applied. Implemented as the GHOST algorithm this approach is capable of handling the huge solution space and provides an insight into the "quasicontinuous" distribution of parameters that describe the biophysical properties of an experimental system. However, the analysis procedure requires several hundreds of runs of the evolutionary optimization routine making this algorithm extremely computationally demanding. As only the best parameter sets from each run are assumed to contribute into the final solution, this algorithm appears far from being optimized. The goal of this study is to modify the optimization routine in a way that 20-40 runs would be enough to obtain qualitatively the same characterization. However, to keep the solution diversity throughout the HEO run, fitness sharing and newly developed shaking mechanisms are applied and tested on various test EPR spectra. In addition, other evolutionary optimization parameters such as population size and probability of genetic operators were also varied to tune the algorithm. According to the testing examples a speed-up factor of 5-7 was achieved.

Algorithms↗

PubMedDate not supplied

Determination of partition coefficient of spin probe between different lipid membrane phases.

Model lipid membranes made from binary mixtures of dimyristoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DMPC/DPPC) and dimyristoylphosphatidylcholine/cholesterol (DMPC/Chol) exhibit coexistence of diverse lipid phases at appropriate temperature and composition. Since lipids in different phases show different structural and motional properties, it is expected that the corresponding spin probe electron paramagnetic resonance (EPR) spectra will be superposition of several spectral components. From comparison of proportions of spectral components of the EPR spectrum with the fractions of the corresponding lipid phases obtained from known phase diagrams the partition coefficient of spin probe methyl ester of 5-doxyl palmitate between different lipid phases was determined. The results indicate that the used spin probe partitions approximately equally between different phases.

1,2-Dipalmitoylphosphatidylcholine↗