A fehérjemátrix szerepe a redoxfehérjék működésében = Role of the protein matrix in the function of redox proteins

Rekombináns citokróm c mutánsokat állítottunk elő és a bevezetett ciszteineket TUPS fotoaktív redox jelölővel jelöltük. Ugyancsak jelöltünk felszíni lizineket. Lézerimpulzussal való gerjesztés után kinetikus spektroszkópiai módszerrel mértük és értelmeztük az elektrontranszfer sebességét a jelölő és a citokróm hem kofaktora között. Megállapítottuk, hogy a jelölő flexibilis kovalens kapcsolata miatt több pozíciót tud felvenni és ezért az elektrontranszfer több exponenciálisból álló kinetikát mutat. A kísérleti adatokat összehasonlítottuk modellszámításokkal és megállapítottuk, hogy mind a konkrét elektrontranszfer útvonalakkal, mind pedig a fehérjével, mint változó sűrűségű folytonos közeggel számoló modell az eredményekkel jól megegyező sebességeket jósol. A citokróm felszínét feltérképeztük elektrontranszfer hatékonyság szempontjából: a hem síkjához viszonyított poláros régiók valamivel hatékonyabbak az egyenlítői régióknál. A citokróm c és a citokróm oxidáz komplexében mérve az elektrontranszfert az eddigieknél nagyobb időfelbontással tudtuk megmérni a CuA (első) elektronakceptor redukciójának idejét, és azt 100 ns-nál gyorsabbnak találtuk. Megállapítottuk, hogy E. coli baktériumban érett holocitokróm c kis mennyiségben spontán módon, hem liáz segítsége nélkül is képződik. Sikerült a hem liáz fehérjét heterológ módon kifejezni és tisztítani, és így apocitokróm és hem felhasználásával in vitro holocitokrómot termelni. | We produced recombinant mutant cytochrome c proteins and labeled the introduced cysteines and native lysines with the photoactive redox label TUPS. Pulse laser excitation of TUPS and the ensuing electron transfer between the label and the heme of cytochrome c was followed by kinetic absorption spectroscopy. Due to the flexible covalent link between the label and the protein TUPS can assume multiple positions which result in multiexponential electron transfer kinetics. We compared the experimental data with model calculations and concluded that both the model assuming explicite electron transfer pathways and the model assuming a protein matrix with varying packing density can satisfactorily describe the experimental observations. We mapped the surface of cytochrome c in terms of electron transfer efficiency towards the heme. The polar regions were found to be slightly more efficient ('hotter') than the equatorial regions relative to the plane of the heme. By measuring the electron transfer in the complex of cytochrome c and cytochrome c oxidase at a higher time resolution than in previous studies, we obtained a reduction time of the primary electron acceptor, CuA, which is faster than 100 ns. We found that in E. coli a low efficiency spontaneous maturation of holocytochrome c can take place without the assistance of heme lyase. We expressed and purified yeast heme lyase from E. coli and demonstrated in vitro holocytochrome maturation upon addition of apocytochrome and heme

Quantitative linear dichroism imaging of molecular processes in living cells made simple by open software tools

Fluorescence-detected linear dichroism microscopy allows observing various molecular processes in living cells, as well as obtaining quantitative information on orientation of fluorescent molecules associated with cellular features. Such information can provide insights into protein structure, aid in development of genetically encoded probes, and allow determinations of lipid membrane properties. However, quantitating and interpreting linear dichroism in biological systems has been laborious and unreliable. Here we present a set of open source ImageJ-based software tools that allow fast and easy linear dichroism visualization and quantitation, as well as extraction of quantitative information on molecular orientations, even in living systems. The tools were tested on model synthetic lipid vesicles and applied to a variety of biological systems, including observations of conformational changes during G-protein signaling in living cells, using fluorescent proteins. Our results show that our tools and model systems are applicable to a wide range of molecules and polarization-resolved microscopy techniques, and represent a significant step towards making polarization microscopy a mainstream tool of biological imaging.</p

Effect of Hofmeister cosolutes on the photocycle of photoactive yellow protein at moderately alkaline pH

The photocycle of photoactive yellow protein was studied by kinetic absorption spectroscopy from below 100 ns to seconds, at moderately alkaline pH, in the presence of high concentrations of various salts. Chemometric analysis combined with multiexponential fit of the flash-induced difference spectra provided evidence for five intermediates, including a spectrally silent form before the final recovery of the parent state, but only three with significantly distinct spectra. The calculated intermediate spectra constituted the input for the following spectrotemporal model fit using a sufficiently complex photocycle scheme with reversible transitions. This yielded the rate coefficients of the molecular transitions, the final spectra and the kinetics of the intermediates. Except for the transition between the two red shifted (early) intermediates (pR(1) and pR(2)) and the final photocycle step, all reactions appeared to be reversible. Kosmotropic and chaotropic cosolutes had a systematic effect on the molecular rate coefficients. The largest effect, associated presumably with the exposure of the hydrophobic interior of the protein, accompanies the transition between the second red-shifted and the first blue-shifted intermediate (pR(2) and pB(1) respectively), i.e. it coincides with the chromophore protonation. The dependence of the rate coefficients on the Hofmeister cosolutes suggests that the conformational change of photoactive yellow protein leading eventually to the most unfolded signaling state takes place in several steps, and starts already with the relaxation after the chromophore isomerization in the microsecond time domain. (C) 2013 Elsevier B.V. All rights reserved

Changes of the absorption cross section of Si nanocrystals with temperature and distance

The absorption cross section (ACS) of silicon nanocrystals (Si NCs) in single-layer and multilayer structures with variable thickness of oxide barriers is determined via a photoluminescence (PL) modulation technique that is based on the analysis of excitation intensity-dependent PL kinetics under modulated pumping. We clearly demonstrate that roughly doubling the barrier thickness (from ca. 1 to 2.2 nm) induces a decrease of the ACS by a factor of 1.5. An optimum separation barrier thickness of ca. 1.6 nm is calculated to maximize the PL intensity yield. This large variation of ACS values with barrier thickness is attributed to a modulation of either defect population states or of the efficiency of energy transfer between confined NC layers. An exponential decrease of the ACS with decreasing temperature down to 120 K can be explained by smaller occupation number of phonons and expansion of the band gap of Si NCs at low temperatures. This study clearly shows that the ACS of Si NCs cannot be considered as independent on experimental conditions and sample parameters