Protein structural change at the cytoplasmic surface as the cause of cooperativity in the bacteriorhodopsin photocycle.

The effects of excitation light intensity on the kinetics of the bacteriorhodopsin photocycle were investigated. The earlier reported intensity-dependent changes at 410 and 570 nm are explained by parallel increases in two of the rate constants, for proton transfers to D96 from the Schiff base and from the cytoplasmic surface, without changes in the others, as the photoexcited fraction is increased. Thus, it appears that the pKa of D96 is raised by a cooperative effect within the purple membrane. This interpretation of the wild-type kinetics was confirmed by results with several mutant proteins, where the rates are well separated in time and a model-dependent analysis is unnecessary. Based on earlier results that demonstrated a structural change of the protein after deprotonation of the Schiff base that increases the area of the cytoplasmic surface, and the effects of high hydrostatic pressure and lowered water activity on the photocycle steps in question, we suggest that the pKa of D96 is raised by a lateral pressure that develops when other bacteriorhodopsin molecules are photoexcited within the two-dimensional lattice of the purple membrane. Expulsion of no more than a few water molecules bound near D96 by this pressure would account for the calculated increase of 0.6 units in the pKa.     

Time-resolved refractive index change during the bacteriorhodopsin photocycle

Kinetic refractive index spectroscopy has been applied to the study of the bacteriorhodopsin photocycle. A fully hydrated purple membrane film was examined in the temperature range from 10° to 40°C using 532 nm excitation (doubled Nd YAG laser) and 633 nm (He–Ne laser) testing beam. Multiexponential fitting of the data revealed five processes. Four of them are well known from kinetic optical absorption studies. The fifth process has only recently been observed in optical absorption experiments where it has a relatively small amplitude. In our refractive index experiments it has an amplitude of up to 30% of the full signal amplitude. It is characterized by an Arrhenius temperature dependence with an activation enthalpy of 40±5 kJ/mol and a decay time of about 0.8 ms at 20°C.     

Conformational change during photocycle of bacteriorhodopsin and its proton-pumping mechanism

Based on the recent finding on the structural difference of seven helix bundles in the all-trans and 13-cis bacteriorhodopsins, the distances among the key groups performing the function of proton translocation as well as their microenvironments have been investigated. Consequently, a pore-gated model was proposed for the light-driven proton-pumping mechanism of bacteriorhodopsin. According to this model, the five double-bounded polyene chain in retinal chromophore can be phenomenologically likened to a molecular “lever,” whose one end links to a “piston” (the β-ionone ring) and the other end to a pump “relay station” (the Schiff base). During the photocycle of bacteriorhodopsin, the molecular “lever” is moving up and down as marked by the position change of the “piston,” so as to trigger the gate of pore to open and close alternately. When the “piston” is up, the pore-controlled gate is open so that the water channel from Asp-96 to the Schiff base and that from the Schiff base to Asp-85 is established; when the “piston” is down, the pore-controlled gate is closed and the water channels for proton transportation in both the cytoplasmic half and extracellular half are blocked. The current model allows a consistent interpretation of a great deal of experimental data and also provides a useful basis for further investigating the mechanism of proton pumping by bacteriorhodopsin.     

Conformational change of helix G in the bacteriorhodopsin photocycle: investigation with heavy atom labeling and x-ray diffraction.

According to the current structural model of bacteriorhodopsin, Ile222 is located at the cytoplasmic end of helix G. We labeled the single cysteine of the site-directed mutant Ile222 --> Cys with p-chloromercuribenzoic acid and determined the position of the labeled mercury by x-ray diffraction in the unphotolyzed state, and in the MN photointermediate accumulated in the presence of guanidine hydrochloride at pH 9.5. According to the difference Fourier maps between the MN intermediate and the unphotolyzed state, the structural change in the MN intermediate was not affected by mercury labeling. The difference Fourier map between the labeled and the unlabeled I222C gave the position of the mercury label. This information was obtained for both the unphotolyzed state and the MN intermediate. We found that the position of the mercury at residue 222 is shifted by 2.1 +/- 0.8 A in the MN intermediate. This agrees with earlier results that suggested a structural change in the G helix. The movement of the mercury label is so large that it must originate from a cooperative conformational change in the helix G at its cytoplasmic end, rather than from displacement of residue 222. Because Ile222 is located at the same level on the z coordinate as Asp96, the structural change in the G helix could have the functional role of perturbing the environment and therefore the pKa of this functionally important aspartate.     

The effect of protein conformation change from alpha(II) to alpha(I) on the bacteriorhodopsin photocycle

The bacteriorhodopsin (bR) photocycle was followed by use of time-resolved Fourier-transform infrared (FTIR) spectroscopy as a function of temperature (15-85 degrees C) as the alpha(II) --> alpha(I) conformational transition occurs. The photocycle rate increases with increasing temperature, but its efficiency is found to be drastically reduced as the transition takes place. A large shift is observed in the all-trans left arrow over right arrow 13-cis equilibrium due to the increased stability of the 13-cis isomer in alpha(I) form. This, together with the increase in the rate of dark adaptation as the temperature increases, leads to a large increase in the 13-cis isomer concentration in bR in the alpha(I) form. The fact that 13-cis retinal has a much-reduced absorption cross-section and its inability to pump protons leads to an observed large reduction in the concentration of the observed photocycle intermediates, as well as the proton gradient at a given light intensity. These results suggest that nature might have selected the alpha(II) rather than the alpha(I) form as the helical conformation in bR to stabilize the all-trans retinal isomer that is a better light absorber and is capable of pumping protons.     

Changes in hydrogen bonding and environment of tryptophan residues on helix F of bacteriorhodopsin during the photocycle: a time-resolved ultraviolet resonance Raman study

Protein structural changes during the photocycle of bacteriorhodopsin were examined by time-resolved ultraviolet resonance Raman (UVRR) spectroscopy. Most of the 244-nm UVRR difference signals of Trp were assigned to either Trp182 or Trp189 using the Trp182 --> Phe and Trp189 --> Phe mutants. The W17 mode of Trp182 shows a wavenumber downshift in the M(1) --> M(2) transition, indicating an increase in hydrogen bonding strength at the indole nitrogen. On the other hand, Trp189 shows Raman intensity increases of the W16 and W18 modes ascribable to an increased hydrophobic interaction. These observations suggest that the tilt of helix F, which ensures that reprotonation of the Schiff base is from the cytoplasmic side, occurs in the M(1) --> M(2) transition. In the M(2) --> N transition, the environment of Trp189 returns to the initial state, whereas the hydrophobic interaction of Trp182 decreases drastically. The decrease in hydrophobic interaction of Trp182 in the N state suggests an invasion of water molecules that promote the proton transfer from Asp96 to the Schiff base. Structural reorganization of the protein after the tilt of helix F may be important for efficient reprotonation of the Schiff base.     

Chromophore reorientation during the photocycle of bacteriorhodopsin: experimental methods and functional significance

Light-induced isomerization leads to orientational changes of the retinylidene chromophore of bacteriorhodopsin in its binding pocket. The chromophore reorientation has been characterized by the following methods: polarized absorption spectroscopy in the visible, UV and IR; polarized resonance Raman scattering; solid-state deuterium nuclear magnetic resonance; neutron and X-ray diffraction. Most of these experiments were performed at low temperatures with bacteriorhodopsin trapped in one or a mixture of intermediates. Time-resolved measurements at room temperature with bacteriorhodopsin in aqueous suspension can currently only be carried out with transient polarized absorption spectroscopy in the visible. The results obtained to date for the initial state and the K, L and M intermediates are presented and discussed. The most extensive data are available for the M intermediate, which plays an essential role in the function of bacteriorhodopsin. For this intermediate the various methods lead to a consistent picture: the curved all-trans polyene chain in the initial state straightens out in the M intermediate (13-cis) and the chain segment between C(5) and C(13) tilts upwards in the direction of the cytoplasmic surface. The kink at C(13) allows the positions of beta-ionone ring and Schiff base nitrogen to remain approximately fixed.     

Distortions in the photocycle of bacteriorhodopsin at moderate dehydration.

The photoreaction of bacteriorhodopsin was studied in moderately dehydrated films (relative humidities between 100 and 65%). Time-resolved difference spectra from a gated optical multichannel analyzer, between 100 ns and 100 ms after photoexcitation, were decomposed into sums of difference spectra of the intermediates K, L, M, N, and O, and the kinetics obtained were fitted to various alternative schemes. The data confirm the model of a single reaction sequence with reversible reactions we proposed recently for purple membrane suspensions (Váró, G., and J. K. Lanyi. Biochemistry. 1990. 29:2241-2250) but including reversibility also for the reaction K in equilibrium with L in addition to L in equilibrium with M, M in equilibrium with N, and N in equilibrium with O. With increasing dehydration the kinetics were increasingly dominated by the reverse reactions. As before, fitting the data required the existence of two M species in series: L in equilibrium with M1 in equilibrium with M2 in equilibrium with N. The M1 in equilibrium with M2 reaction was greatly slowed at lower humidities. This step might be the switch for the unidirectional transfer of protons. With increasing dehydration recovery of BR occurred less and less via the N intermediate and increasingly via direct shunts from the two M species. As indicated earlier by electrical measurements with similarly dried bacteriorhodopsin films (Váró, G., and L. Keszthelyi, 1983. Biophys. J. 43:47-51). The latter are pathways not necessarily associated with net proton translocation.     

Functional roles of aspartic acid residues at the cytoplasmic surface of bacteriorhodopsin.

The functions of the four aspartic acid residues in interhelical loops at the cytoplasmic surface of bacteriorhodopsin, Asp-36, Asp-38, Asp-102, and Asp-104, were investigated by studying single and multiple aspartic acid to asparagine mutants. The same mutants were examined also with the additional D96N residue replacement. The kinetics of the M and N intermediates of the photochemical cycles of these recombinant proteins were affected only in a minor, although self-consistent, way. When residue 38 is an aspartate and anionic, it makes the internal proton exchange between the retinal Schiff base and Asp-96 about 3 times more rapid, and events associated with the reisomerization of retinal to all-trans about 3 times slower. Asp-36 has the opposite effect on these processes, but to a smaller extent. Asp-102 and Asp-104 have even less or none of these effects. Of the four aspartates, only Asp-36 could play a direct role in proton uptake at the cytoplasmic surface. In the 13 bacterioopsin sequences now available, only this surface aspartate is conserved.     

Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.     

Electric signals during the bacteriorhodopsin photocycle, determined over a wide pH range.

From the electric signals measured after photoexcitation, the electrogenicity of the photocycle intermediates of bacteriorhodopsin were determined in a pH range of 4.5-9. Current measurements and absorption kinetic signals at five wavelengths were recorded in the time interval from 300 ns to 0.5 s. To fit the data, the model containing sequential intermediates connected by reversible first-order reactions was used. The electrogenicities were calculated from the integral of the current signal, by using the time-dependent concentrations of the intermediates, obtained from the fits. Almost all of the calculated electrogenicities were pH independent, suggesting that the charge motions occur inside the protein. Only the N intermediate exhibited pH-dependent electrogenicity, implying that the protonation of Asp96, from the intracellular part of the protein, is not from a well-determined proton donor. The calculated electrogenicities gave good approximations of all of the details of the measured electric signals.     

Functional roles of tyrosine 185 during the bacteriorhodopsin photocycle as revealed by in situ spectroscopic studies

Tyrosine 185 (Y185), one of the aromatic residues within the retinal (Ret) chromophore binding pocket in helix F of bacteriorhodopsin (bR), is highly conserved among the microbial rhodopsin family proteins. Many studies have investigated the functions of Y185, but its underlying mechanism during the bR photocycle remains unclear. To address this research gap, in situ two-dimensional (2D) magic-angle spinning (MAS) solid-state NMR (ssNMR) of specifically labelled bR, combined with light-induced transient absorption change measurements, dynamic light scattering (DLS) measurements, titration analysis and site-directed mutagenesis, was used to elucidate the functional roles of Y185 during the bR photocycle in the native membrane environment. Different interaction modes were identified between Y185 and the Ret chromophore in the dark-adapted (inactive) state and M (active) state, indicating that Y185 may serve as a rotamer switch maintaining the protein dynamics, and plays an important role in the efficient proton-pumping mechanism in the bR purple membrane.     

Crystallographic analysis of protein conformational changes in the bacteriorhodopsin photocycle

A variety of neutron, X-ray and electron diffraction experiments have established that the transmembrane regions of bacteriorhodopsin undergo significant light-induced changes in conformation during the course of the photocycle. A recent comprehensive electron crystallographic analysis of light-driven structural changes in wild-type bacteriorhodopsin and a number of mutants has established that a single, large protein conformational change occurs within 1 ms after illumination, roughly coincident with the time scale of formation of the M(2) intermediate in the photocycle of wild-type bacteriorhodopsin. Minor differences in structural changes that are observed in mutants that display long-lived M(2), N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. These observations support a model for the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M(1)) are well approximated by one protein conformation in which the Schiff base has extracellular accessibility, while the structures of the later intermediates (M(2), N and O) are well approximated by the other protein conformation in which the Schiff base has cytoplasmic accessibility.     

The quantum efficiency of the bacteriorhodopsin photocycle.

The quantum yield of the primary photoprocess in light-adapted bacteriorhodopsin (phi 1) was determined at room temperature with low-intensity 530 nm neodymium laser excitation, with bovine rhodopsin as a relative actinometer. The observed value of phi 1 - 0.25 +/- 0.05, and the previously determined parameter phi 1/phi 2 - 0.4 [where phi 2 denotes the quantum efficiency of the back photoprecess from the primary species K (590)] imply that phi 1 + phi 2 approximately equal 1. This feature, also characterizing the photochemistry of rhodopsin, bears on the nature and mechanism of the primary event in both systems.     

Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.

A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

     

Electric response of a back photoreaction in the bacteriorhodopsin photocycle.

The electric response of a back photoreaction in the bacteriorhodopsin photocycle was investigated. The proton pumping activity of green flash excited bacteriorhodopsin stops if the M412 form is illuminated by blue light (Karvaly and Dancsházy, 1977). In the present work a fast negative displacement current signal was measured in an oriented membrane suspension system, indicative of back movement of protons from M412 to BR570. Quantitative evaluation of the data shows that there are at least two steps in the back reaction, with different rate constants. The temperature dependence of the rate constants show simple linear Arrhenius behavior between 5 degree and 40 degree C. The rate constants were slower by a factor of 1.8 in D2O suspension. The relevance of the protein electric response signals (PERS) observed in this paper to the early receptor potential is discussed.     

Pathways of proton release in the bacteriorhodopsin photocycle.

The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.     

Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes

The expression of membrane proteins for functional and structural studies or medicinal applications is still not very well established. Membrane-spanning proteins that mediate the information flow of the extracellular side with the interior of the cell are prime targets for drug development methods that would allow screening techniques or high throughput formats are of particular interest. Here we describe a systematic approach to the liposome-assisted cell-free synthesis of functional membrane proteins. We demonstrate the synthesis of bacteriorhodopsin (bR(cf)) in presence of small unilamellar liposomes. The yield of bR(cf) per volume cell culture is comparable to that of bacteriorhodopsin in its native host. The functional analysis of bR(cf) was performed directly using the cell-free reaction mixture. Photocycle measurements reveal kinetic data similar to that determined for bR in Halobacterium salinarum cell-envelope vesicles. The liposomes can be attached directly to black lipid membranes (BLM), which allows measuring light activated photocurrents in situ. The results reveal a functional proton pump with properties identical to those established for the native protein.     

Structural changes during the formation of early intermediates in the bacteriorhodopsin photocycle

Early intermediates of bacteriorhodopsin's photocycle were modeled by means of ab initio quantum mechanical/molecular mechanical and molecular dynamics simulations. The photoisomerization of the retinal chromophore and the formation of photoproducts corresponding to the early intermediates were simulated by molecular dynamics simulations. By means of the quantum mechanical/molecular mechanical method, the resulting structures were refined and the respective excitation energies were calculated. Two sequential intermediates were found with absorption maxima that exhibit red shifts from the resting state. The intermediates were therefore assigned to the K and KL states. In K, the conformation of the retinal chromophore is strongly deformed, and the N--H bond of the Schiff base points almost perpendicular to the membrane normal toward Asp-212. The strongly deformed conformation of the chromophore and weakened interaction of the Schiff base with the surrounding polar groups are the means by which the absorbed energy is stored. During the K-to-KL transition, the chromophore undergoes further conformational changes that result in the formation of a hydrogen bond between the N--H group of the Schiff base and Thr-89 as well as other rearrangements of the hydrogen-bond network in the vicinity of the Schiff base, which are suggested to play a key role in the proton transfer process in the later phase of the photocycle.