Igor Chizhov

Proteorhodopsin is a Light-driven Proton Pump with Variable Vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions. The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.

Spectrally silent transitions in the bacteriorhodopsin photocycle

The photocycle kinetics of bacteriorhodopsin were analyzed from 0 to 40 degrees C at 101 wavelengths (330-730 nm). The data can be satisfactorily approximated by eight exponents. The slowest component (half-time 20 ms at 20 degrees C) belongs to the 13-cis cycle. The residual seven exponentials that are sufficient to describe the all-trans photocycle indicate that at least seven intermediates of the all-trans cycle must exist, although only five spectrally distinct species (K, L, M, N, and O) have been identified. These seven exponentials and their spectra at different temperatures provide the basis for the discussion of various kinetic schemes of the relaxation. The simplest model of irreversible sequential transitions includes after the first K--> L step the quasiequilibria of L<-->M, M<-->N, and N<-->O intermediates. These quasiequilibria are controlled by rate-limiting dynamics of the protein and/or proton transfer steps outside the chromophore region. Thus there exists an apparent kinetic paradox (i.e., why is the number of exponents of relaxation (at least seven) higher than the number of distinct spectral intermediates (only five)), which can be explained by assuming that some of the transitions correspond to changes in the quasiequilibria between spectrally distinct intermediates (i.e., are spectrally silent).

Cofilin cooperates with fascin to disassemble filopodial actin filaments

Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.

Sensory Rhodopsin II from the Haloalkaliphilic Natronobacterium pharaonis: Light-Activated Proton Transfer Reactions

In the present work the light-activated proton transfer reactions of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) and those of the channel-mutants D75N-pSRII and F86D-pSRII are investigated using flash photolysis and black lipid membrane (BLM) techniques. Whereas the photocycle of the F86D-pSRII mutant is quite similar to that of the wild-type protein, the photocycle of D75N-pSRII consists of only two intermediates. The addition of external proton donors such as azide, or in the case of F86D-pSRII, imidazole, accelerates the reprotonation of the Schiff base, but not the turnover. The electrical measurements prove that pSRII and F86D-pSRII can function as outwardly directed proton pumps, whereas the mutation in the extracellular channel (D75N-pSRII) leads to an inwardly directed transient current. The almost negligible size of the photostationary current is explained by the long-lasting photocycle of about a second. Although the M decay, but not the photocycle turnover, of pSRII and F86D-pSRII is accelerated by the addition of azide, the photostationary current is considerably increased. It is discussed that in a two-photon process a late intermediate (N- and/or O-like species) is photoconverted back to the original resting state; thereby the long photocycle is cut short, giving rise to the large increase of the photostationary current. The results presented in this work indicate that the function to generate ion gradients across membranes is a general property of archaeal rhodopsins.

Microbial Rhodopsins: Scaffolds for Ion Pumps, Channels, and Sensors

Microbial rhodopsins have been intensively researched for the last three decades. Since the discovery of bacteriorhodopsin, the scope of microbial rhodopsins has been considerably extended, not only in view of the large number of family members, but also their functional properties as pumps, sensors, and channels. In this review, we give a short overview of old and newly discovered microbial rhodopsins, the mechanism of signal transfer and ion transfer, and we discuss structural and mechanistic aspects of phototaxis.

Static and Time-Resolved Step-Scan Fourier Transform Infrared Investigations of the Photoreaction of Halorhodopsin from Natronobacterium Pharaonis: Consequences for Models of the Anion Translocation Mechanism

The molecular changes during the photoreaction of halorhodopsin from Natronobacterium pharaonis have been monitored by low-temperature static and by time-resolved step-scan Fourier transform infrared difference spectroscopy. In the low-temperature L spectrum anions only influence a band around 1650 cm(-1), tentatively assigned to the C=N stretch of the protonated Schiff base of L. The analysis of the time-resolved spectra allows to identify the four states: K, L(1), L(2), and O. Between L(1) and L(2), only the apoprotein undergoes alterations. The O state is characterized by an all-trans chromophore and by rather large amide I spectral changes. Because in our analysis the intermediate containing O is in equilibrium with a state indistinguishable from L(2), we are unable to identify an N-like state. At very high chloride concentrations (>5 M), we observe a branching of the photocycle from L(2) directly back to the dark state, and we provide evidence for direct back-isomerization from L(2). This branching leads to the reported reduction of transport activity at such high chloride concentrations. We interpret the L(1) to L(2) transition as an accessibility change of the anion from the extracellular to the cytosolic side, and the large amide I bands in O as an indication for opening of the cytosolic channel from the Schiff base toward the cytosolic surface and/or as indication for changes of the binding constant of the release site.

KINETIC MODEL OF BACTERIORHODOPSIN PHOTOCYCLE: PATHWAY FROM M STATE TO bR

Abstract

Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

A flash photolysis fluorescence/light scattering apparatus for use with sub microgram quantities of muscle proteins

Transient kinetic methods such as stopped flow and quenched flow have been used to elucidate many of the fundamental features of the molecular interactions which underlie muscle contraction. However, these methods traditionally require relatively large amounts of protein (10−3 g) and so have been used most effectively for the proteins purified from bulk muscle tissue of large animals or where the proteins can be expressed in large amounts (e.g., Dictyostelium). We have investigated the use of flash photolysis of an inert precursor of ATP (cATP) to initiate the dissociation of acto.S1 and acto.myosin and the subsequent ATP turnover reaction. Using a sample volume of 10 μl we show that a significant amount of information on the transient and steady-state kinetics of the system can be obtained from a sample containing just 50 nM of acto.myosin or acto.S1 complex in solution. Therefore in presence of excess of one protein component the measurements require only 250 ng myosin, 62 ng S1 or 25 ng actin. This is therefore the method of choice for kinetic analysis of acto.myosins which are only available in microgram quantities. We report for the first time the determination of the second order rate constant of ATP-induced dissociation of actin from the myosin extracted from a single fibre from a rabbit psoas muscle.

Dictyostelium Myosin-5b Is a Conditional Processive Motor

Dictyostelium myosin-5b is the gene product of myoJ and one of two closely related myosin-5 isoenzymes produced in Dictyostelium discoideum. Here we report a detailed investigation of the kinetic and functional properties of the protein. In standard assay buffer conditions, Dictyostelium myosin-5b displays high actin affinity in the presence of ADP, fast ATP hydrolysis, and a high steady-state ATPase activity in the presence of actin that is rate limited by ADP release. These properties are typical for a processive motor that can move over long distances along actin filaments without dissociating. Our results show that a physiological decrease in the concentration of free Mg2+-ions leads to an increased rate of ADP release and shortening of the fraction of time the motor spends in the strong actin binding states. Consistently, the ability of the motor to efficiently translocate actin filaments at very low surface densities decreases with decreasing concentrations of free Mg2+-ions. In addition, we provide evidence that the observed changes in Dd myosin-5b motor activity are of physiological relevance and propose a mechanism by which this molecular motor can switch between processive and non-processive movement.