High-resolution structural insights into the heliorhodopsin family.

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR's extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.

Pressure dependence of the photocycle kinetics of bacteriorhodopsin.

The pressure dependence of the photocycle kinetics of bacteriorhodopsin from Halobacterium salinarium was investigated at pressures up to 4 kbar at 25 degrees C and 40 degrees C. The kinetics can be adequately modeled by nine apparent rate constants, which are assigned to irreversible transitions of a single relaxation chain of nine kinetically distinguishable states P(1) to P(9). All states except P(1) and P(9) consist of two or more spectral components. The kinetic states P(2) to P(6) comprise only the two fast equilibrating spectral states L and M. From the pressure dependence, the volume differences DeltaV(o)(LM) between these two spectral states could be determined that range from DeltaV(o)(LM) = -11.4 +/- 0.7 ml/mol (P(2)) to DeltaV(o)(LM) = 14.6 +/- 2.8 mL/mol (P(6)). A model is developed that explains the dependence of DeltaV(o)(LM) on the kinetic state by the electrostriction effect of charges, which are formed and neutralized during the L/M transition.

Temperature and halide dependence of the photocycle of halorhodopsin from Natronobacterium pharaonis.

The photocycle kinetics of halorhodopsin from Natronobacterium pharaonis (pHR(575)) was analyzed at different temperatures and chloride concentrations as well as various halides. Over the whole range of modified parameters the kinetics can be adequately modeled with six apparent rate constants. Assuming a model in which the observed rates are assigned to irreversible transitions of a single relaxation chain, six kinetically distinguishable states (P(1-6)) are discernible that are formed from four chromophore states (spectral archetypes S(j): K(570), L(N)(520), O(600), pHR'(575)). Whereas P(1) coincides with K(570) (S(1)), both P(2) and P(3) have identical spectra resembling L(520) (S(2)), thus representing a true spectral silent transition between them. P(4) constitutes a fast temperature-dependent equilibrium between the chromophore states S(2) and S(3) (L(520) and O(600), respectively). The subsequent equilibrium (P(5)) of the same spectral archetypes is only moderately temperature dependent but shows sensitivity toward the type of anion and the chloride concentration. Therefore, S(2) and S(3) occurring in P(4) as well as in P(5) have to be distinguished and are assigned to L(520)<--> O(1)(600) and O(2)(600)<--> N(520) equilibrium, respectively. It is proposed that P(4) and P(5) represent the anion release and uptake steps. Based on the experimental data affinities of the halide binding sites are estimated.

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.

Studies of electron transport dynamics in photosynthetic reaction centers using fast temperature changes.

Rates of thermoinduced conformational transitions of reaction center (RC) complexes providing effective electron transport were studied in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria using methods of fast freezing and laser-induced temperature jump. Reactions of electron transfer from the primary to secondary quinone acceptors and from the multiheme cytochrome c subunit to photoactive bacteriochlorophyll dimer were used as probes of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. In contrast to the quinone complex, the thermoinduced transition of the macromolecular RC complex to the state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220-280 K accounts for tens of seconds. This transition is thought to be mediated by large-scale conformational dynamics of the macromolecular RC complex.

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 microl 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.

Time-resolved detection of transient movement of helix F in spin-labelled pharaonis sensory rhodopsin II.

Sensory rhodopsin II (also called phoborhodopsin) from the archaeal Natronobacterium pharaonis (pSRII) functions as a repellent phototaxis receptor. The excitation of the receptor by light triggers the activation of a transducer molecule (pHtrII) which has close resemblance to the cytoplasmic domain of bacterial chemotaxis receptors. In order to elucidate the first step of the signal transduction chain, the accessibility as well as static and transient mobility of cytoplasmic residues in helices F and G were analysed by electron paramagnetic resonance spectroscopy. The results indicate an outward tilting of helix F during the early steps of the photocycle which is sustained until the reformation of the initial ground state. Co-expression of pSRII with a truncated fragment of pHtrII affects the accessibility and/or the mobility of certain spin-labelled residues on helices F and G. The results suggest that these sites are located within the binding surface of the photoreceptor with its transducer.

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.

Functional expression of His-tagged sensory rhodopsin I in Escherichia coli.

Sensory rhodopsin I (SRI) from Halobacterium salinarum was functionally expressed in Escherichia coli and subsequently purified to homogeneity using a C-terminal His-tag anchor. Yields of 3-4 mg SRI/l cell culture can be obtained. The absorption and photocycle properties of SRI were similar if not indistinguishable from those of the homologously expressed SRI. A global fit analysis of the photocycle data and the calculation of the spectra of states provided strong evidence for the existence of an N-like intermediate.

Time-resolved step-scan Fourier transform infrared spectroscopy reveals differences between early and late M intermediates of bacteriorhodopsin.

In this report, from time-resolved step-scan Fourier transform infrared investigations from 15 ns to 160 ms, we provide evidence for the subsequent rise of three different M states that differ in their structures. The first state rises with approximately 3 microseconds to only a small percentage. Its structure as judged from amide I/II bands differs in small but well-defined aspects from the L state. The next M state, which appears in approximately 40 microseconds, has almost all of the characteristics of the "late" M state, i.e., it differs considerably from the first one. Here, the L left arrow over right arrow M equilibrium is shifted toward M, although some percentage of L still persists. In the last M state (rise time approximately 130 microseconds), the equilibrium is shifted toward full deprotonation of the Schiff base, and only small additional structural changes take place. In addition to these results obtained for unbuffered conditions or at pH 7, experiments performed at lower and higher pH are presented. These results are discussed in terms of the molecular changes postulated to occur in the M intermediate to allow the shift of the L/M equilibrium toward M and possibly to regulate the change of the accessibility of the Schiff base necessary for effective proton pumping.

The photophobic receptor from Natronobacterium pharaonis: temperature and pH dependencies of the photocycle of sensory rhodopsin II.

The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H2O with D2O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states Si correspond in their spectral properties to those of the BR photocycle, namely pSRII510 (K), pSRII495 (L), pSRII400 (M), pSRII485 (N), and pSRII535 (O). In comparison to BR, pSRII400 is formed approximately 10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of DeltaS. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H2O/D2O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.

The use of fast kinetic systems for the study of ng-microgram quantities of purified 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 (several mg) 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 been developing methods which would allow fast transients to be studied on the much smaller quantities of protein available from individual drosophila muscles, single mammalian muscle fibres, human biopsies and non-muscle myosins. The use of fluorescent labels on actin, myosin or ATP can be used in modern stopped-flow equipment at concentrations as low as 10 nM. These labels can report on the interactions between the three molecules using a few microgram of protein. However the stopped flow systems requires large volumes of sample to give sufficient acceleration of the sample to achieve good mixing. We have therefore been using a similar optical set up but initiating the reaction using caged ATP. In this device the same measurements can be made using 10-20 fold less material. This is the quantity of myosin which can be purified from a few mm of a single mammalian muscle fibre. Using these methods the following measurements can be made: rate of ATP induced dissociation of actomyosin, the affinity of ADP for actomyosin, the rate of ADP release from actomyosin and the affinity of actin for myosin and myosin ADP.

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).

Temperature and pH sensitivity of the O(640) intermediate of the bacteriorhodopsin photocycle.

The temperature and pH dependencies of the O(640) intermediate of the photocycle of bacteriorhodopsin (bR) were investigated by flash photolysis and T-jump experiments. The maximal concentration of the O(640) intermediate was found to be dependent on the temperature, which is described by a sigmoidal relationship. With increasing pH the midpoint of the sigmoidal curves shifts to higher temperatures. The Van't Hoff equation provides enthalpy and entropy values of the observed states. These results indicate that, in the investigated temperature (0-60 degrees C) and pH (pH 4.0-10.0) range, the sequence of the principal intermediates in the pathway "M-N-O-bR" does not change. The observations of the O(640) intermediate at pH < 8.0 and of the N(550) intermediate at pH > 8.0 are most probably due only to changes of the intrinsic rate constants of the bR photocycle, not to a different mechanism.

Kinetic model of bacteriorhodopsin photocycle: pathway from M state to bR.

A model of the last parts of the bacteriorhodopsin (bR) photocycle is proposed on the basis of experimental data for the kinetic behavior of the 'O' intermediate during a temperature pulse in distilled water suspension. The model includes the previously proposed (but not well characterized) intermediate 'N' between the 'M' and 'O' states of bR. This intermediate exists in fast temperature-dependent quasi-stationary equilibrium with the red-shifted intermediate 'O' and has a maximum of absorption close to the bR spectrum.

[Kinetics of primary photocycle stages of bacteriorhodopsin at low temperatures]. / Kinetika pervichnykh stadii fototsikla bakteriorodopsina pri nizkikh temperaturakh.

According to the changes of absorption spectra kinetics of two primary stages of bacteriorhodopsin photochemical cycle was studied in the temperature range 160 +/- 300 degrees K. It has been found that for K610-L550 transition in the range under study the rate-temperature relationship is described by Arrhenius law with the activation energy Ea = 0.68 eV. For L550-M412 transition Ea = 0.69 eV. The character of temperature relationship, of the rate and amplitude for this transition indicates that at T less than or equal to 270 degrees K a phase transition is possible.