Elucidation of the viral disassembly switch of tobacco mosaic virus.

Stable capsid structures of viruses protect viral RNA while they also require controlled disassembly for releasing the viral genome in the host cell. A detailed understanding of viral disassembly processes and the involved structural switches is still lacking. This process has been extensively studied using tobacco mosaic virus (TMV), and carboxylate interactions are assumed to play a critical part in this process. Here, we present two cryo-EM structures of the helical TMV assembly at 2.0 and 1.9 Å resolution in conditions of high Ca2+ concentration at low pH and in water. Based on our atomic models, we identify the conformational details of the disassembly switch mechanism: In high Ca2+ /acidic pH environment, the virion is stabilized between neighboring subunits through carboxyl groups E95 and E97 in close proximity to a Ca2+ binding site that is shared between two subunits. Upon increase in pH and lower Ca2+ levels, mutual repulsion of the E95/E97 pair and Ca2+ removal destabilize the network of interactions between adjacent subunits at lower radius and release the switch for viral disassembly.

Development of a Thermofluor assay for stability determination of membrane proteins using the Na(+)/H(+) antiporter NhaA and cytochrome c oxidase.

Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochrome c oxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex.

True wild type and recombinant wild type cytochrome c oxidase from Paracoccus denitrificans show a 20-fold difference in their catalase activity.

The four subunit (SU) aa(3) cytochrome c oxidase (CcO) from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain. Its binuclear active center, residing in SU I, contains heme a(3) and Cu(B). Apart from its oxygen reductase activity, the protein possesses a peroxidase and a catalase activity. To compare variants and the wild type (WT) protein in a more stringent way, a recombinant (rec.) WT strain was constructed, carrying the gene for SU I on a low copy number plasmid. This rec. WT showed no difference in oxygen reductase activity compared to the American Type Culture Collection (ATCC) WT CcO but surprisingly its catalase activity was increased by a factor of 20. The potential over-production of SU I might impair the correct insertion of heme a(3) and Cu(B) because of a deficiency in metal inserting chaperones. An altered distance between heme a(3) and Cu(B) and variations in protein structure are possible reasons for the observed increased catalase activity. The availability of chaperones was improved by cloning the genes ctaG and surf1c on the same plasmid as the SU I gene. The new rec. WT CcO showed in fact a reduced catalase activity. Using differential scanning calorimetry no significant difference in thermal stability between the ATCC WT CcO and the rec. WT CcO was detected. However, upon aging the thermal stability of the rec. WT CcO was reduced compared to that of the ATCC WT CcO pointing to a decreased structural stability of the rec. WT CcO.

Evidence for distinct electron transfer processes in terminal oxidases from different origin by means of protein film voltammetry.

Cytochrome aa3 from Paracoccus denitrificans and cytochrome ba3 from Thermus thermophilus, two distinct members of the heme-copper oxidase superfamily, were immobilized on electrodes modified with gold nanoparticles. This procedure allowed us to achieve direct electron transfer between the enzyme and the gold nanoparticles and to obtain evidence for different electrocatalytic properties of the two enzymes. The pH dependence and thermostability reveal that the enzymes are highly adapted to their native environments. These results suggest that evolution resulted in different solutions to the common problem of electron transfer to oxygen.

Interconversions of P and F intermediates of cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. This redox-driven proton pump catalyzes the four-electron reduction of molecular oxygen to water, one of the most fundamental processes in biology. Elucidation of the intermediate structures in the catalytic cycle is crucial for understanding both the mechanism of oxygen reduction and its coupling to proton pumping. Using CcO from Paracoccus denitrificans, we demonstrate that the artificial F state, classically generated by reaction with an excess of hydrogen peroxide, can be converted into a new P state (in contradiction to the conventional direction of the catalytic cycle) by addition of ammonia at pH 9. We suggest that ammonia coordinates directly to Cu(B) in the binuclear active center in this P state and discuss the chemical structures of both oxoferryl intermediates F and P. Our results are compatible with a superoxide bound to Cu(B) in the F state.

Resonance Raman characterization of the ammonia-generated oxo intermediate of cytochrome c oxidase from Paracoccus denitrificans.

A novel oxo state of cytochrome c oxidase from Paracoccus denitrificans generated by successive addition of excess H2O2 and ammonia was investigated using resonance Raman (RR) spectroscopy. Addition of ammonia to the H2O2-generated artificial F state resulted in an upshift of the oxoferryl stretching vibration from 790 to 796 cm(-1), indicating that ammonia influences ligation of the heme-bound oxygen in the binuclear center. Concomitantly performed RR measurements in the high-frequency region between 1300 and 1700 cm(-1) showed a high-spin to low-spin transition of heme a3 upon generation of the F state that was not altered by addition of ammonia. Removal of H2O2 by addition of catalase resulted in the disappearance of the oxoferryl stretching vibration and major back transformation of heme a3 into the high-spin state. The ratio of high-spin to low-spin states was identical for intermediates created with and without ammonia, leading to the conclusion that ammonia does not interact directly with heme a3. Only for the ammonia-created state was a band at 612 nm observed in the UV-visible difference spectrum that was shifted to 608 nm after addition of catalase. Our results support the hypothesis by von der Hocht et al. [von der Hocht, I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 3964-3969] that addition of ammonia creates a novel oxo intermediate state called PN where ammonia binds to CuB once the oxo intermediate F state has been formed.

Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS.

In order to monitor membrane-protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual-focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two-dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D = 2.2 x 10(-8) cm2 s(-1) for the photoreceptor and with D = 4.1 x 10(-8) cm2 s(-1) for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecule's cylindrical radius, we are able to estimate the degree of membrane protein binding on a quantitative level.

Measuring large numerical apertures by imaging the angular distribution of radiation of fluorescing molecules.

Exact knowledge of the numerical aperture is crucial in many applications using high-aperture objectives such as confocal microscopy, optical trapping, or advanced sub-wavelength imaging methods. We propose and apply a precise and straightforward method for measuring this fundamental parameter of microscope objectives with numerical apertures above unity. Our method exploits the peculiarities of the fluorescence emission of molecules at a glass/air interface.

Fluorescence correlation spectroscopy in cells: confinement and excluded volume effects.

Fluorescence correlation spectroscopy (FCS) has become an important technique in biophysical research, which is also used for in vivo studies of molecular mobilities in cells. We theoretically study how confinement or exclusion of the diffusing fluorescent molecules by a spherical region influences the measured autocorrelation function in an FCS experiment. It is shown that close to the boundary of the spherical region the diffusion time can be significantly changed due to the geometric restriction of the detection volume. This is important when quantitatively evaluating and interpreting FCS measurements in cells.

Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements.

We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two-parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two-focus-FCS, the diffusion coefficient of 4.26 x 10(-6) cm2s(-1) for Atto655 in water at 25 degrees C compares well with the GPF NMR value of 4.28 x 10(-6) cm2s(-1).

Surface sticking and lateral diffusion of lipids in supported bilayers.

The diffusion of fluorescently labeled lipids in supported bilayers is studied using two different methods: Z-scan fluorescence correlation spectroscopy (z-scan FCS) and two-focus fluorescence correlation spectroscopy (2f-FCS). It is found that the data can be fitted consistently only when taking into account partial sticking of the labeled lipids to the supporting glass surface. A kinetic reaction-diffusion model is developed and applied to the data. We find a very slow sticking rate which, however, when neglected, leads to strongly varying estimates of the free diffusion coefficient. The study reveals a strong sensitivity of FCS on even slight binding/unbinding kinetics of the labeled molecules, which has significance for related diffusion measurements in cellular lipid membranes.