In this paper a new atomic force sensing technique is presented for dynamically probing conformational changes in proteins. The method is applied to the light-induced changes in the membrane-bound proton pump bacteriorhodopsin (bR). The microsecond time-resolution of the method, as presently implemented, covers many of the intermediates of the bR photocycle which is well characterized by spectroscopical methods. In addition to the native pigment, we have studied bR proteins substituted with chemically modified retinal chromophores. These synthetic chromophores were designed to restrict their ability to isomerize, while maintaining the basic characteristic of a large light-induced charge redistribution in the vertically excited Franck–Condon state. An analysis of the atomic force sensing signals lead us to conclude that protein conformational changes in bR can be initiated as a result of a light-triggered redistribution of electronic charge in the retinal chromophore, even when isomerization cannot take place. Although the coupling mechanism of such changes to the light-induced proton pump is still not established, our data question the current working hypothesis which attributes all primary events in retinal proteins to an initial trans ⇔ cis isomerization.
We have applied low temperature difference FTIR spectroscopy to investigate intermediates produced from the M intermediate upon blue light excitation (<480 nm). In agreement with an earlier report by Balashov and Litvin (1981), who studied these intermediates with low temperature visible absorption spectrophotometry, we have observed at least three stages in this backphotoreaction. The initial photoproduct is stable at 100 K, and two products of subsequent thermal reactions are observed upon raising the temperature to 130 and 160 K, respectively.The alterations in the C=N stretching mode of the Schiff base have been identified by isotopically labeling the retinal chromophore, and changes in C=O stretching modes of amino acid residues with acidic side chains have been investigated. Analysis of the C=N stretching mode shows that the Schiff base remains unprotonated after the photochemical reaction at 100 K. Moreover, there are two types of Schiff bases, presumably associated with different bR species, that become thermally reprotonated at 130 and 160 K, respectively. Bands associated with the C=O stretching modes suggest that Asp 85 rather than Asp 96 reprotonates the Schiff base during the M to bR backphotoreaction. This conclusion is consistent with earlier observations that the polarity of electrical signals during this photochemical back reaction is reversed as compared to the thermal regeneration of bR from M.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA mechanism for controlling the pKa of the retinal protonated Schiff base in retinal proteins. A study with model compoundsY. Gat and M. ShevesCite this: J. Am. Chem. Soc. 1993, 115, 9, 3772–3773Publication Date (Print):May 1, 1993Publication History Published online1 May 2002Published inissue 1 May 1993https://pubs.acs.org/doi/10.1021/ja00062a052https://doi.org/10.1021/ja00062a052research-articleACS PublicationsRequest reuse permissionsArticle Views520Altmetric-Citations89LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTOn the Heterogeneity of the M Population in the Photocycle of BacteriorhodopsinN. Friedman, Y. Gat, M. Sheves, and M. OttolenghiCite this: Biochemistry 1994, 33, 49, 14758–14767Publication Date (Print):December 1, 1994Publication History Published online1 May 2002Published inissue 1 December 1994https://pubs.acs.org/doi/10.1021/bi00253a014https://doi.org/10.1021/bi00253a014research-articleACS PublicationsRequest reuse permissionsArticle Views57Altmetric-Citations19LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
The primary light-induced events in the photosynthetic retinal protein bacteriorhodopsin (bR) are investigated by ultrafast optical spectroscopy over the 440−1000 nm spectral range. The study compares the early dynamics of the native all-trans pigment bR570 with those of two synthetic analogues, bR5.12 and bR5.13, in which isomerization around the critical C13C14 bond is blocked by a five-membered ring into all-trans and 13-cis configurations, respectively. Nearly identical spectral evolution is observed in both native and artificial systems over the first 100−200 fs of probe delay. During this period stimulated near-IR (∼900 nm) emission, and intense ∼460 nm absorption bands, due to analogous fluorescent I states (denoted as I460, I5.12 and I5.13, respectively), appear concurrently within 30 fs. In all systems continuous spectral shifting over tens of femtoseconds is observed in the 500−700 nm range. Native bR goes on to produce the J625 absorption band within ∼1 ps, which is accompanied by disappearance of the I460 emission and absorption features. In bR5.12 and bR5.13, aside from minor spectral modifications, the analogous dramatic changes associated with I5.12 and I5.13 are arrested beyond the first ∼100 fs, reverting uniformly to the initial ground state with exponential time constants of 19 ps and 11 ps, respectively. Analysis of the data calls for a major revision of models previously put forward for the primary events in bacteriorhodopsin. The close likeness of initial transient spectral evolution in both native and artificial pigments, despite the locking of the active isomerization coordinate in the synthetic chromophores, demonstrates that in bR570 the ultrafast changes in transmission leading to I460, previously believed to involve C13C14 torsion, must be associated with other modes. The detailed comparison conducted here also identifies which of the later spectral changes in the native system requires torsional flexibility in C13C14. Similarity of 660 nm probing data in both synthetic and native chromophores demonstrates that the sub-picosecond dynamic features uncovered at this probing wavelength commonly attributed to the evolution of J625, are not, as previously thought, reliable measures of all-trans ⇔ 13-cis isomerization dynamics.
Upon light adaptation by continuous (or pulsed) illumination, the artificial bacteriorhodopsin (bR) pigments, I and II, derived from synthetic 14F retinal and a short polyenal, respectively produce a long-lived red-shifted species denoted Ol. An analogous phenomenon was observed by Sonar, S., et al. [(1993) Biochemistry 32, 2263-2271], in the case of the Y185F mutant (pigment III). The nature of these Ol species was investigated by studying a series of effects, primarily their red light photoreversibility, the associated proton uptake and release processes, and the effects of pH on their relative amounts, which are interpreted in terms of pH-dependent acid−base equilibria. Experiments were also carried out with pigments I and II derived from the mutants D96A, E204Q, R82Q, and D85N. The Ol species of pigments I and II (and possibly also that of pigment III) are identified as an unusually long-lived (all-trans) intermediate of the photocycle of their 13-cis isomer. It is concluded that in Ol, Asp-85 is protonated, a process associated with proton uptake from the extracellular side. Subsequent proton release (to the same side of the membrane) occurs from Glu-204 (or from a group closely interacting with it) prior to the decay of Ol. At high pH (>9), Ol reversibly converts to a purple form, due to deprotonation of Asp-85, while at still higher pH (>11), a blue-shifted species characterized by a deprotonated Schiff base is generated. These transitions constitute the first demonstration of the titration of a photocycle intermediate of a retinal protein. The respective pKa values are determined and discussed in relation to those pertaining to the unphotolyzed (dark-adapted) pigments. It appears that the pKa values are controlled by a hydrogen bond network involving water molecules, which binds the protonated Schiff base with Asp-85 and Glu-204. The disruption of this network in pigments I-III may also be responsible for the long lifetime of the Ol species, due to the inhibition of thermal trans−13-cis isomerization. The results are relevant to the molecular mechanism of the photocycles of both 13-cis- and all-trans-bR, primarily to the nature and to the deprotonation mechanism of the proton-releasing group.
Bacteriorhodopsin (bR) has been biosynthetically prepared with lysine deuterated at its alpha carbon (C alpha--H). The labeled membranes containing bR were investigated by difference Fourier transform infrared (FTIR) spectroscopy. It has been derived from K/bR and M/bR difference spectra (K and M are photocycle intermediates) that several bands previously assigned to the retinal chromophore are coupled to the C alpha--H. The vibrational modes that exhibit this coupling are principally associated with C15--H and N--H vibrations. [C alpha--2H]Lysine-labeled bR was fragmented enzymatically, and bR structures were regenerated with the C alpha--2H label either on lysine-216 and -172 or on the remaining five lysine residues of the protein. FTIR studies of the regenerated bR system, together with methylation of all lysines except the active-site lysine, reveal that the changes observed due to backbone labeling arise from the active-site lysine. The intensity of the C15--H out-of-plane wag is interpreted as a possible indication of a twist around the C15 = N bond.
Abstract The factors that red shift the absorption maximum of the retinal Schiff base chromophore in the M 412 intermediate of bacteriorhodopsin photocycle relative to absorption in solution were investigated using a series of artificial pigments and studies of model compounds in solution. The artificial pigments derived from retinal analogs that perturb chromophore‐protein interactions in the vicinity of the ring moiety indicate that a considerable part of the red shift may originate from interactions in the vicinity of the Schiff base linkage. Studies with model compounds revealed that hydrogen bonding to the Schiff base moiety can significantly red shift the absorption maximum. Furthermore, it was demonstrated that although s‐trans ring‐chain planarity prevails in the M 412 intermediate it does not contribute significantly (only ca 750 cm −1 ) to the opsin shift observed in M 412 . It is suggested that in M 412 , the Schiff base linkage is hydrogen bonded to bound water and/or protein residues inducing a considerable red shift in the absorption maximum of the retinal chromophore.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe Retinal Schiff Base-Counterion Complex of Bacteriorhodopsin: Changed Geometry during the Photocycle Is a Cause of Proton Transfer to Aspartate 85Leonid S. Brown, Yahaloma Gat, Mordechai Sheves, Yoichi Yamazaki, Akio Maeda, Richard Needleman, and Janos K. LanyiCite this: Biochemistry 1994, 33, 40, 12001–12011Publication Date (Print):October 11, 1994Publication History Published online1 May 2002Published inissue 11 October 1994https://pubs.acs.org/doi/10.1021/bi00206a001https://doi.org/10.1021/bi00206a001research-articleACS PublicationsRequest reuse permissionsArticle Views227Altmetric-Citations48LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
