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.
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Effect of nitrification on defixation of clay fixed ammonium was studied in soil with pronounced ammonium fixing capacity. Soil samples were amended with 15N labeled ammonium. Significant part of ...added ammonium (50–70 %) was not exchangeable with 2N KCl after the first 10 minutes of incubation. Dynamics of 15N in 2N KCl exchangeable mineral nitrogen was followed during 28 days of aerobic and anaerobic incubation. Exchangeable ammonium was microbialy converted to nitrite and nitrate during aerobic incubation. When the exchangeable ammonium pool was empty, a slow defixation of chemically fixed ammonium occurred. Approximately 20 % of additionally fixed ammonium was released in the second half of aerobic incubation. No defixation occurred during anaerobic incubation.
Vpliv nitrifikacije na defiksacijo amonijskega dušika smo proučevali v talnem vzorcu z visoko kapaciteto za fiksacijo dušika. Izbrana tla so vezala 50-70 % dodanega amonijskega dušika v obliko, ki po 10 minutah ni bila več izmenljiva z 2N KCl. Dinamiko z 15N označenega mineralnega dušika smo opazovali v laboratorijskem modelnem sistemu med 28 dnevno aerobno in anaerobno inkubacijo. V aerobnih razmerah je bil 2N KCl izmenljivi amonijski dušik mikrobno presnovljen do nitrita in nitrata. V drugi polovici aerobne inkubacije, ko je bila zaloga izmenljivega amonija porabljena, se je sprostilo v 2N KCl izmenljivo obliko približno 20 % fiksiranega 15N-amonijskega dušika. Pri anaerobni inkubaciji ni prišlo do sproščanja fiksiranega amonija.
Substrate-induced respiration (SIR) was used to estimate microbial respiration and microbial biomass in soils from Stavešinci natural CO2 spring (mofette) exposed to different geological CO2 ...concentrations. SIR measurements clearly demonstrated higher microbial respiration and microbial biomass in control sites compared to high soil CO2 sites. Sampling in two different locations and in three different years also confirmed long-term stability of this pattern, which was found for both locations and in different sampling periods.
Bakterije se na različite načine prilagođavaju promjenama uvjeta okoline. U ovom su kratkom preglednom radu opisane različite strategije prilagodbe crveno pigmentirane bakterije Vibrio ruber, nedavno ...izolirane iz priobalja, na čimbenike stresa (tj. salinitet, viskoznost, UV svjetlost, mitomicin C, pristupačnost hranjiva i temperaturu). Bakterija Vibrio ruber se koristi različitim strategijama adaptacije kako bi se oduprla okolišnom stresu. Ovisno o koncentraciji soli, bakterija Vibrio ruber mijenja svoj lipidni sastav, te svojstva lipidne faze. Membrana se bakterije Vibrio ruber razlikuje od ostalih srodnih vrsta bakterije Vibrio po tome što ne sadržava hidroksi masne kiseline, ali zato ima izrazito velik udjel lizolipida. Nakupljanje anorganskih hranjivih tvari u bakteriji je selektivno i ovisi o uvjetima okoline. Bakterije se brzo prilagođavaju stresnim uvjetima i mijenjaju svoj proteinski sastav, metabolizam, tj. potrošnju ugljika i energije, te proizvodnju sekundarnih metabolita. Aktivnost glukoza-6-fosfat dehidrogenaze dobar je indikator stresa u Vibrio ruber. Bakterije mogu mijenjati viskozitet stanica kao odgovor na promjenu viskoziteta okoline. Imaju nekoliko virusnih elemenata u genomu koji se mogu inducirati mitomicinom C. Promjene u uvjetima okoline tijekom rasta bakterija bitno utječu na iskorištenje ugljika iz lizata mikrobnih stanica. Nedavno je otkrivena nova ekofiziološka funkcija sekundarnog metabolita prodigiozina, a to je da štiti stanicu od UV zračenja. Može se zaključiti da se u kratkom periodu istraživanja bakterije Vibrio ruber (kraćem od deset godina) dokazalo da se može upotrijebiti kao vrlo učinkovit model za ispitivanje ekofiziologije bakterija.
The local environment of the transmembrane and C-terminal domain of M13 major coat protein was probed by site-directed ESR spin labeling when the protein was introduced into three membrane-mimicking ...systems, DOPC vesicles, sodium cholate micelles, and SDS micelles. For this purpose, we have inserted unique cysteine residues at specific positions in the transmembrane and C-terminal region, using site-directed mutagenesis. Seven viable mutants with reasonable yield were harvested: A25C, V31C, T36C, G38C, T46C, A49C, and S50C. The mutant coat proteins were indistinguishable from wild type M13 coat protein with respect to their conformational and aggregational properties. The ESR data suggest that the amino acid positions 25 and 46 of the coat protein in DOPC vesicles are located close to the membrane−water interface. In this way the lysines at positions 40, 43, and 44 and the phenylalanines at positions 42 and 45 act as hydrophilic and hydrophobic anchors, respectively. The ESR spectra of site specific maleimido spin-labeled mutant coat proteins reconstituted into DOPC vesicles and solubilized in sodium cholate or SDS indicate that the local dynamics of the major coat protein is significantly affected by its structural environment (micellar vs bilayer), location (aqueous vs hydrophobic), and lipid/protein ratio. The detergents SDS and sodium cholate sufficiently well solubilize the major coat protein and largely retain its secondary structure elements. However, the results indicate that they have a poorly defined protein-amphiphilic structure and lipid−water interface as compared to bilayers and thus are not a good substitute for lipid bilayers in biophysical studies.
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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.
INTRODUCTIONDifferent people have different views of what modeling biological phenomena is all about. For some, models are mathematical equations, also known as formal models (Haefner, 1996), that ...represent, for example, the growth of populations. For others, complex systems are studied in a simplified form in the laboratory, using experiments as physical models (Haefner, 1996). In other words, models, as broadly defined, are simplified representations of reality. This simplifying of the real world, though crucial to modeling success, nevertheless is a dangerous practice. Simplify too far, and a model's solutions, whether mathematical or empirical, will have nothing to do with reality. Simplify too little, and the model becomes too difficult to solve or too complex to derive useful conclusions from. A typical and ecologically very legitimate simplification, for example, is to ignore molecular or physiological details to concentrate instead on properties of whole organisms such as their fecundity or age of death. In this tradition, here we consider phage—bacterial, predator—prey interactions in fluid environments.The environment of phages and bacteria may be modeled as unchanging over time, changing due to phage or bacterial actions (e.g., bacterial depletion due to phage growth or substrate consumption due to bacterial growth), or changing as a consequence of factors external to both phages and bacteria (e.g., due to chemostat outflow or protist grazing).
The structure and changes in environment of the M13 major coat protein were studied in model systems, mimicking the initial molecular process of the phage disassembly. For this purpose we have ...systematically studied protein associations with various detergents and lipids in two different coat protein assemblies: phage particles and S-forms. It is remarkable that the major coat protein can change its conformation to accommodate three distinctly different environments: phage filament, S-form, and membrane-bound form. The structural and environmental changes during this protein transformations were studied by site-directed spin labeling, fluorescence labeling, and CD spectroscopy in different membrane model systems. The phage particles were disrupted only by strong ionic detergents sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide and (CTAB) but were not affected by sodium cholate and sodium deoxycholate, nonionic detergents, and dilauroyl-l-α-phosphatidylcholine (DLPC) lipid bilayers. Conversion of the phage particles into S-forms by addition of chloroform rendered the coat protein accessible for the association with different ionic and nonionic detergents, as well as DLPC lipids. The disruption of the S-form by all detergents studied was instantaneous but was slower with DLPC vesicles. Only small unilamellar vesicles effectively solubilized the S-form. The data suggest that the viral protein coat is inherently unstable when the major coat protein is exposed to amphiphilic molecules. During conversion from the phage to the S-form, and subsequently to the membrane-bound form, the coat protein undergoes pronounced changes in environment, and in response the α-helix content decreases and the local protein structure changes dramatically. This adaptation of the protein conformation enables a stable association of the protein with the membrane.
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