Photoactivation of the BLUF protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues

Handle URI:
http://hdl.handle.net/10754/625460
Title:
Photoactivation of the BLUF protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues
Authors:
Gil, Agnieszka A.; Laptenok, Sergey P.; Iuliano, James N.; Lukacs, Andras; Verma, Anil; Hall, Christopher R.; Yoon, EunBin; Brust, Richard; Greetham, Gregory M.; Towrie, Michael; French, Jarrod B.; Meech, Stephen R.; Tonge, Peter J
Abstract:
The flavin chromophore in blue light using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppA by the introduction of fluorotyrosine (F-Tyr) analogs that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark to light adapted form) photoreaction was observed, the change in Y21 pKa led to a 4,000-fold increase in the rate of dark state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppA, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppA, despite their sharing highly conserved FAD binding architectures.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Citation:
Gil AA, Laptenok SP, Iuliano JN, Lukacs A, Verma A, et al. (2017) Photoactivation of the BLUF protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues. Journal of the American Chemical Society. Available: http://dx.doi.org/10.1021/jacs.7b07849.
Publisher:
American Chemical Society (ACS)
Journal:
Journal of the American Chemical Society
Issue Date:
6-Sep-2017
DOI:
10.1021/jacs.7b07849
Type:
Article
ISSN:
0002-7863; 1520-5126
Sponsors:
Funded by the EPSRC (EP/G002916 to SRM) and NSF (CHE-1223819 to PJT). We are grateful to STFC for access to the ULTRA laser facility. We are grateful to Professor Ray Owens for assistance in protein preparation and access to the Oxford Protein Production Facility – UK, and to Arthur Makarenko at CSHL for assistance with the mass spectrometry analysis. JI was supported by an NIH Chemistry-Biology Interface training grant (T32GM092714). AL is a Bolyai Janos Research Fellow and was supported by OTKA NN113090.
Additional Links:
http://pubs.acs.org/doi/abs/10.1021/jacs.7b07849
Appears in Collections:
Articles; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorGil, Agnieszka A.en
dc.contributor.authorLaptenok, Sergey P.en
dc.contributor.authorIuliano, James N.en
dc.contributor.authorLukacs, Andrasen
dc.contributor.authorVerma, Anilen
dc.contributor.authorHall, Christopher R.en
dc.contributor.authorYoon, EunBinen
dc.contributor.authorBrust, Richarden
dc.contributor.authorGreetham, Gregory M.en
dc.contributor.authorTowrie, Michaelen
dc.contributor.authorFrench, Jarrod B.en
dc.contributor.authorMeech, Stephen R.en
dc.contributor.authorTonge, Peter Jen
dc.date.accessioned2017-09-14T06:03:52Z-
dc.date.available2017-09-14T06:03:52Z-
dc.date.issued2017-09-06en
dc.identifier.citationGil AA, Laptenok SP, Iuliano JN, Lukacs A, Verma A, et al. (2017) Photoactivation of the BLUF protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues. Journal of the American Chemical Society. Available: http://dx.doi.org/10.1021/jacs.7b07849.en
dc.identifier.issn0002-7863en
dc.identifier.issn1520-5126en
dc.identifier.doi10.1021/jacs.7b07849en
dc.identifier.urihttp://hdl.handle.net/10754/625460-
dc.description.abstractThe flavin chromophore in blue light using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppA by the introduction of fluorotyrosine (F-Tyr) analogs that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark to light adapted form) photoreaction was observed, the change in Y21 pKa led to a 4,000-fold increase in the rate of dark state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppA, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppA, despite their sharing highly conserved FAD binding architectures.en
dc.description.sponsorshipFunded by the EPSRC (EP/G002916 to SRM) and NSF (CHE-1223819 to PJT). We are grateful to STFC for access to the ULTRA laser facility. We are grateful to Professor Ray Owens for assistance in protein preparation and access to the Oxford Protein Production Facility – UK, and to Arthur Makarenko at CSHL for assistance with the mass spectrometry analysis. JI was supported by an NIH Chemistry-Biology Interface training grant (T32GM092714). AL is a Bolyai Janos Research Fellow and was supported by OTKA NN113090.en
dc.publisherAmerican Chemical Society (ACS)en
dc.relation.urlhttp://pubs.acs.org/doi/abs/10.1021/jacs.7b07849en
dc.rightsThis document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/jacs.7b07849.en
dc.titlePhotoactivation of the BLUF protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residuesen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.identifier.journalJournal of the American Chemical Societyen
dc.eprint.versionPost-printen
dc.contributor.institutionDepartment of Chemistryen
dc.contributor.institutionSchool of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UKen
dc.contributor.institutionDepartment of Biophysics, Medical School, University of Pecs, Pecs, Hungaryen
dc.contributor.institutionCentral Laser Facility, Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX, UK.en
dc.contributor.institutionBiochemistry & Cell Biology, Stony Brook University, Stony Brook, New York 11794-3400, USAen
kaust.authorLaptenok, Sergey P.en
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