The active site of an ion pump must communicate alternately with the two opposite membrane surfac... more The active site of an ion pump must communicate alternately with the two opposite membrane surfaces. In the light-driven proton pump, bacteriorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the acceptor of the proton of D96 (with access to the cytoplasmic side). This "reprotonation switch" has been associated with a protein conformation change observed during the photocycle. When D85 is replaced with asparagine, the pKa value of the Schiff base is lowered from above 13 to about 9. We determined the direction of the loss or gain of the Schiff base proton in unphotolyzed and in photoexcited D85N, and the D85N/D96N and D85N/D96A double mutants, in order to understand the intrinsic and the induced connectivities of the Schiff base to the two membrane surfaces. The influence of D96 mutations on proton exchange and on acceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to the cytoplasmic side. In the wild-type protein (but with the pKa of the Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the extracellular side. Raising the pH without illumination of D85N so as to deprotonate the Schiff base caused the same, or nearly the same, change of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site to the global protein conformation and to the connectivity of the Schiff base proton to the membrane surfaces. Their relationship suggests that the conformation of the unphotolyzed wild-type protein is stabilized by coulombic interaction of the Schiff base with its counter-ion. A proton is translocated across the membrane after light-induced transfer of the Schiff base proton to D85, because the protein assumes an alternative conformation that separates the donor from the acceptor and opens new conduction pathways between the active site and the two membrane surfaces.
Leptosphaeria rhodopsin (LR) is a light-driven proton pumping retinal protein found in Leptosphae... more Leptosphaeria rhodopsin (LR) is a light-driven proton pumping retinal protein found in Leptosphaeria maculans, a eukaryotic organism. LR pumps protons in a manner similar to bacteriorhodopsin (BR), a light-driven proton pump of halobacteria. The protein structure around the retinal chromophore in LR and its structural change upon retinal photoisomerization resemble those in BR. The photocycle intermediates of LR resemble the K, L, M, N and O states of BR. The electric signals provided information about the charge motion during the photocycle. There is one basic difference between the electric signals of BR and LR: the positive part typical for BR could not be detected in the electrical signal of LR.
Photonic Applications in Nonlinear Optics, Nanophotonics, and Microwave Photonics, 2005
The all-trans to 13-cis isomerization of the retinal chromophore in bacteriorhodopsin (bR) plays ... more The all-trans to 13-cis isomerization of the retinal chromophore in bacteriorhodopsin (bR) plays an essential role in Nature (e.g., in photosynthesis of halobacteria). bR is a candidate for optical nanodevices driven by laser pulses, and a prospective material for optical memory storage devices and photoswitches. From the viewpoint of possible applications of bR in nanodevices we performed an experimental study
Since the discovery of proteorhodopsins, the ubiquitous marine light-driven proton pumps of eubac... more Since the discovery of proteorhodopsins, the ubiquitous marine light-driven proton pumps of eubacteria, a large number of other eubacterial rhodopsins with diverse structures and functions have been characterized. Here, we review the body of knowledge accumulated on the four major groups of eubacterial rhodopsins, with the focus on their biophysical characterization. We discuss advances and controversies on the unique eubacterial sensory rhodopsins (as represented by Anabaena sensory rhodopsin), proton-pumping proteorhodopsins and xanthorhodopsins, as well as novel non-proton ion pumps. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.
Proceedings of the National Academy of Sciences, 2005
Bacteriorhodopsin-like proteins provide archaea and eubacteria with a unique bioenergetic pathway... more Bacteriorhodopsin-like proteins provide archaea and eubacteria with a unique bioenergetic pathway comprising light-driven transmembrane proton translocation by a single retinal-binding protein. Recently, homologous proteins were found to perform photosensory functions in lower eukaryotes, but no active ion transport by eukaryotic rhodopsins was detected. By demonstrating light-driven proton pumping in a fungal rhodopsin from Leptosphaeria maculans, we present a case of a retinal-based proton transporter from a eukaryote. This result implies that in addition to oxidative phosphorylation and chlorophyll photosynthesis, some lower eukaryotes may have retained the archaeal route of building an electrochemical transmembrane gradient of protons.
Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) ar... more Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D(2) O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N-H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR.
We have measured proton release into the medium after proton transfer from the retinal Schiff bas... more We have measured proton release into the medium after proton transfer from the retinal Schiff base to Asp85 in the photocycle and the C = O stretch bands of carboxylic acids in wild type bacteriorhodopsin and the E204Q and E204D mutants. In E204Q, but not in E204D, the normal proton release is absent. Consistent with this, a negative band in the Fourier transform infrared difference spectra at 1700 cm-1 in the wild type, which we now attribute to depletion of the protonated E204, is also absent in E204Q. In E204D, this band is shifted to 1714 cm-1, as expected from the higher frequency for a protonated aspartic than for a glutamic acid. Consistent with their origin from protonated carboxyls, the depletion bands in the wild type and E204D shift in D2O to 1690 and 1703 cm-1, respectively. In the protein structure, Glu204 seems to be connected to the Schiff base region by a chain of hydrogen-bonded water. As with other residues closer to the Schiff base, replacement of Glu204 with glutamine changes the O-H stretch frequency of the bound water molecule near Asp85 that undergoes hydrogen-bonding change in the photocycle. The results therefore identify Glu204 as XH, the earlier postulated residue that is the source of the released proton during the transport, and suggest that its deprotonation is triggered by the protonation of Asp85 through a network that contains water dipoles.
The chromophores of the D85T and D85N mutants of bacteriorhodopsin are blue but become purple lik... more The chromophores of the D85T and D85N mutants of bacteriorhodopsin are blue but become purple like the wild type when chloride or bromide binds near the Schiff base. In D85T this occurs near neutral pH, but in D85N only at pH < 4. The structures of the L and the unphotolyzed states of these proteins were examined with Fourier transform infrared spectroscopy. The difference spectra of the purple forms, but not the blue forms in the absence of these anions, resembled the spectrum of the wild-type protein. Shift of the ethylenic band toward lower frequency upon replacing chloride by bromide confirmed the contribution of the negative charge of the anions to the Schiff base counterion. These anions restored the change of water, which is bound near the protonated Schiff base but is absent in the blue form of the D85N mutant, though with stronger H-bonding than in the wild type. The C = N stretching vibration of the Schiff base in H2O and 2H2O was detected by Fourier transform Raman spectroscopy. The H-bonding strength of the Schiff base in the unphotolyzed state was weaker when chloride or bromide was bound to the mutants than with Asp85 as the counterion in the wild type. Thus, although the geometry of the environment is different, there is at least one water molecule coordinated to the bound halide in these mutants, in a way similar to water bound to Asp85 in the wild type.
Leptosphaeria rhodopsin (LR) is an archaeal-type rhodopsin found in fungi, and is the first light... more Leptosphaeria rhodopsin (LR) is an archaeal-type rhodopsin found in fungi, and is the first light-driven proton-pumping retinal protein from eukaryotes. LR pumps protons in a manner similar to that of bacteriorhodopsin (BR), a light-driven proton pump of haloarchaea. The amino acid sequence of LR is more homologous to that of Neurospora rhodopsin (NR) than BR, whereas NR has no proton-pumping activity. These facts raise the question of how the proton-pumping function is achieved. In this paper, we studied structural changes of LR following the retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy, and compared the obtained spectra with those for BR and NR. While the light-induced photoisomerization from the all-trans to 13-cis form was commonly observed among LR, BR, and NR, we found that the structural changes of LR are closer to those of BR than to those of NR in terms of detailed vibrational bands of retinal and protein. The most prominent difference was seen for the water O-D stretching vibrations (measured in D2O). LR exhibits an O-D stretch of water at 2257 cm(-1), indicating the presence of a strongly hydrogen-bonded water molecule. Such strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) were observed for BR, but not for NR. Comprehensive studies of BR mutants and archaeal rhodopsins have revealed that strongly hydrogen-bonded water molecules are found only in the proteins exhibiting proton-pumping activity, suggesting that strongly hydrogen-bonded water molecules and transient weakening of their binding are essential for the proton-pumping function of rhodopsins. This observation for LR provided additional experimental evidence of the correlation between strongly hydrogen-bonded water molecules and proton-pumping activity of archaeal rhodopsins.
An intense indole N-H stretching vibrational band at 3486 cm-1 in the difference Fourier transfor... more An intense indole N-H stretching vibrational band at 3486 cm-1 in the difference Fourier transform infrared spectrum is one of the characteristic features of the L intermediate of bacteriorhodopsin [Maeda, Sasaki, Ohkita, Simpson, & Herzfeld (1992) Biochemistry 31, 12543]. This band is now assigned to tryptophan-182. The Trp182-->Phe (W182F) protein shows specific features in the difference spectrum in the visible region upon L formation, and exhibits great delay in the L-M conversion. Fourier transform infrared difference spectra further indicate that while the intensity of the C-methyl in-plane bending vibration at 1009 cm-1 is lost in the L intermediate of the wild type, its intensity remains high in the W182F protein. The intensity of the N-H stretching vibration upon L formation is diminished considerably in an artificial bacteriorhodopsin containing 9-desmethylretinal. It also exhibits delayed M formation. These results suggest that Trp182 interacts with the retinal side chain through the 9-methyl group, and thereby affects the L-to-M conversion.
International Conference on Ultrafast Phenomena, 2010
Conflicting results have been obtained between weak field experiments (one-photon absorption) [1]... more Conflicting results have been obtained between weak field experiments (one-photon absorption) [1] and strong field recent studies [2] (multi-photon effects). Here we present our strong field experiments performed using linearly-chirped excitation pulses. Contrary to [2], we ...
International Conference on Ultrafast Phenomena, 2010
1. Introduction Two-dimensional spectroscopy in the photon echo fashion (2D-PE) has been successf... more 1. Introduction Two-dimensional spectroscopy in the photon echo fashion (2D-PE) has been successfully applied for studying structural dynamics and chemical reactions particularly in the IR where the observables are basically the IR-transitions of different components of large ...
In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transpo... more In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982–3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction) is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences
The active site of an ion pump must communicate alternately with the two opposite membrane surfac... more The active site of an ion pump must communicate alternately with the two opposite membrane surfaces. In the light-driven proton pump, bacteriorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the acceptor of the proton of D96 (with access to the cytoplasmic side). This "reprotonation switch" has been associated with a protein conformation change observed during the photocycle. When D85 is replaced with asparagine, the pKa value of the Schiff base is lowered from above 13 to about 9. We determined the direction of the loss or gain of the Schiff base proton in unphotolyzed and in photoexcited D85N, and the D85N/D96N and D85N/D96A double mutants, in order to understand the intrinsic and the induced connectivities of the Schiff base to the two membrane surfaces. The influence of D96 mutations on proton exchange and on acceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to the cytoplasmic side. In the wild-type protein (but with the pKa of the Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the extracellular side. Raising the pH without illumination of D85N so as to deprotonate the Schiff base caused the same, or nearly the same, change of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site to the global protein conformation and to the connectivity of the Schiff base proton to the membrane surfaces. Their relationship suggests that the conformation of the unphotolyzed wild-type protein is stabilized by coulombic interaction of the Schiff base with its counter-ion. A proton is translocated across the membrane after light-induced transfer of the Schiff base proton to D85, because the protein assumes an alternative conformation that separates the donor from the acceptor and opens new conduction pathways between the active site and the two membrane surfaces.
Leptosphaeria rhodopsin (LR) is a light-driven proton pumping retinal protein found in Leptosphae... more Leptosphaeria rhodopsin (LR) is a light-driven proton pumping retinal protein found in Leptosphaeria maculans, a eukaryotic organism. LR pumps protons in a manner similar to bacteriorhodopsin (BR), a light-driven proton pump of halobacteria. The protein structure around the retinal chromophore in LR and its structural change upon retinal photoisomerization resemble those in BR. The photocycle intermediates of LR resemble the K, L, M, N and O states of BR. The electric signals provided information about the charge motion during the photocycle. There is one basic difference between the electric signals of BR and LR: the positive part typical for BR could not be detected in the electrical signal of LR.
Photonic Applications in Nonlinear Optics, Nanophotonics, and Microwave Photonics, 2005
The all-trans to 13-cis isomerization of the retinal chromophore in bacteriorhodopsin (bR) plays ... more The all-trans to 13-cis isomerization of the retinal chromophore in bacteriorhodopsin (bR) plays an essential role in Nature (e.g., in photosynthesis of halobacteria). bR is a candidate for optical nanodevices driven by laser pulses, and a prospective material for optical memory storage devices and photoswitches. From the viewpoint of possible applications of bR in nanodevices we performed an experimental study
Since the discovery of proteorhodopsins, the ubiquitous marine light-driven proton pumps of eubac... more Since the discovery of proteorhodopsins, the ubiquitous marine light-driven proton pumps of eubacteria, a large number of other eubacterial rhodopsins with diverse structures and functions have been characterized. Here, we review the body of knowledge accumulated on the four major groups of eubacterial rhodopsins, with the focus on their biophysical characterization. We discuss advances and controversies on the unique eubacterial sensory rhodopsins (as represented by Anabaena sensory rhodopsin), proton-pumping proteorhodopsins and xanthorhodopsins, as well as novel non-proton ion pumps. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.
Proceedings of the National Academy of Sciences, 2005
Bacteriorhodopsin-like proteins provide archaea and eubacteria with a unique bioenergetic pathway... more Bacteriorhodopsin-like proteins provide archaea and eubacteria with a unique bioenergetic pathway comprising light-driven transmembrane proton translocation by a single retinal-binding protein. Recently, homologous proteins were found to perform photosensory functions in lower eukaryotes, but no active ion transport by eukaryotic rhodopsins was detected. By demonstrating light-driven proton pumping in a fungal rhodopsin from Leptosphaeria maculans, we present a case of a retinal-based proton transporter from a eukaryote. This result implies that in addition to oxidative phosphorylation and chlorophyll photosynthesis, some lower eukaryotes may have retained the archaeal route of building an electrochemical transmembrane gradient of protons.
Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) ar... more Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D(2) O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N-H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR.
We have measured proton release into the medium after proton transfer from the retinal Schiff bas... more We have measured proton release into the medium after proton transfer from the retinal Schiff base to Asp85 in the photocycle and the C = O stretch bands of carboxylic acids in wild type bacteriorhodopsin and the E204Q and E204D mutants. In E204Q, but not in E204D, the normal proton release is absent. Consistent with this, a negative band in the Fourier transform infrared difference spectra at 1700 cm-1 in the wild type, which we now attribute to depletion of the protonated E204, is also absent in E204Q. In E204D, this band is shifted to 1714 cm-1, as expected from the higher frequency for a protonated aspartic than for a glutamic acid. Consistent with their origin from protonated carboxyls, the depletion bands in the wild type and E204D shift in D2O to 1690 and 1703 cm-1, respectively. In the protein structure, Glu204 seems to be connected to the Schiff base region by a chain of hydrogen-bonded water. As with other residues closer to the Schiff base, replacement of Glu204 with glutamine changes the O-H stretch frequency of the bound water molecule near Asp85 that undergoes hydrogen-bonding change in the photocycle. The results therefore identify Glu204 as XH, the earlier postulated residue that is the source of the released proton during the transport, and suggest that its deprotonation is triggered by the protonation of Asp85 through a network that contains water dipoles.
The chromophores of the D85T and D85N mutants of bacteriorhodopsin are blue but become purple lik... more The chromophores of the D85T and D85N mutants of bacteriorhodopsin are blue but become purple like the wild type when chloride or bromide binds near the Schiff base. In D85T this occurs near neutral pH, but in D85N only at pH < 4. The structures of the L and the unphotolyzed states of these proteins were examined with Fourier transform infrared spectroscopy. The difference spectra of the purple forms, but not the blue forms in the absence of these anions, resembled the spectrum of the wild-type protein. Shift of the ethylenic band toward lower frequency upon replacing chloride by bromide confirmed the contribution of the negative charge of the anions to the Schiff base counterion. These anions restored the change of water, which is bound near the protonated Schiff base but is absent in the blue form of the D85N mutant, though with stronger H-bonding than in the wild type. The C = N stretching vibration of the Schiff base in H2O and 2H2O was detected by Fourier transform Raman spectroscopy. The H-bonding strength of the Schiff base in the unphotolyzed state was weaker when chloride or bromide was bound to the mutants than with Asp85 as the counterion in the wild type. Thus, although the geometry of the environment is different, there is at least one water molecule coordinated to the bound halide in these mutants, in a way similar to water bound to Asp85 in the wild type.
Leptosphaeria rhodopsin (LR) is an archaeal-type rhodopsin found in fungi, and is the first light... more Leptosphaeria rhodopsin (LR) is an archaeal-type rhodopsin found in fungi, and is the first light-driven proton-pumping retinal protein from eukaryotes. LR pumps protons in a manner similar to that of bacteriorhodopsin (BR), a light-driven proton pump of haloarchaea. The amino acid sequence of LR is more homologous to that of Neurospora rhodopsin (NR) than BR, whereas NR has no proton-pumping activity. These facts raise the question of how the proton-pumping function is achieved. In this paper, we studied structural changes of LR following the retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy, and compared the obtained spectra with those for BR and NR. While the light-induced photoisomerization from the all-trans to 13-cis form was commonly observed among LR, BR, and NR, we found that the structural changes of LR are closer to those of BR than to those of NR in terms of detailed vibrational bands of retinal and protein. The most prominent difference was seen for the water O-D stretching vibrations (measured in D2O). LR exhibits an O-D stretch of water at 2257 cm(-1), indicating the presence of a strongly hydrogen-bonded water molecule. Such strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) were observed for BR, but not for NR. Comprehensive studies of BR mutants and archaeal rhodopsins have revealed that strongly hydrogen-bonded water molecules are found only in the proteins exhibiting proton-pumping activity, suggesting that strongly hydrogen-bonded water molecules and transient weakening of their binding are essential for the proton-pumping function of rhodopsins. This observation for LR provided additional experimental evidence of the correlation between strongly hydrogen-bonded water molecules and proton-pumping activity of archaeal rhodopsins.
An intense indole N-H stretching vibrational band at 3486 cm-1 in the difference Fourier transfor... more An intense indole N-H stretching vibrational band at 3486 cm-1 in the difference Fourier transform infrared spectrum is one of the characteristic features of the L intermediate of bacteriorhodopsin [Maeda, Sasaki, Ohkita, Simpson, & Herzfeld (1992) Biochemistry 31, 12543]. This band is now assigned to tryptophan-182. The Trp182-->Phe (W182F) protein shows specific features in the difference spectrum in the visible region upon L formation, and exhibits great delay in the L-M conversion. Fourier transform infrared difference spectra further indicate that while the intensity of the C-methyl in-plane bending vibration at 1009 cm-1 is lost in the L intermediate of the wild type, its intensity remains high in the W182F protein. The intensity of the N-H stretching vibration upon L formation is diminished considerably in an artificial bacteriorhodopsin containing 9-desmethylretinal. It also exhibits delayed M formation. These results suggest that Trp182 interacts with the retinal side chain through the 9-methyl group, and thereby affects the L-to-M conversion.
International Conference on Ultrafast Phenomena, 2010
Conflicting results have been obtained between weak field experiments (one-photon absorption) [1]... more Conflicting results have been obtained between weak field experiments (one-photon absorption) [1] and strong field recent studies [2] (multi-photon effects). Here we present our strong field experiments performed using linearly-chirped excitation pulses. Contrary to [2], we ...
International Conference on Ultrafast Phenomena, 2010
1. Introduction Two-dimensional spectroscopy in the photon echo fashion (2D-PE) has been successf... more 1. Introduction Two-dimensional spectroscopy in the photon echo fashion (2D-PE) has been successfully applied for studying structural dynamics and chemical reactions particularly in the IR where the observables are basically the IR-transitions of different components of large ...
In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transpo... more In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982–3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction) is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences
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