title: Time-resolved rapid-scan Fourier transform infrared difference spectroscopy on a noncyclic photosystem: rhodopsin photointermediates from Lumi to Meta II.
abstract: The visual pigment rhodopsin has been extensively studied for the kinetics of its photointermediates by various spectroscopic methods. Unlike such archaeal retinal proteins as bacteriorhodopsin, visual rhodopsin does not thermally recover its dark state after photoexcitation, which precludes repeated excitation of a single sample and thereby complicates time-resolved experiments. Kinetic data on the late rhodopsin photointermediates have so far been available mainly from time-resolved ultraviolet (UV)-visible spectroscopy, but not from Fourier transform infrared (FTIR) spectroscopy. The latter has the advantage of being informative of structural changes of both chromophore and protein, but does not allow the highly reproducible, automated sample exchange procedures available to UV-visible spectroscopy. Using rapid-scan FTIR difference spectroscopy, we obtained time-resolved data sets that were analyzed by a maximum entropy inverse Laplace-transform. Covering the time range from 8 ms to 15 s at temperatures of 0 and -7 degrees C, the transitions from the Lumi to the Meta I and from the Meta I to the Meta II photoproduct states could be resolved. In the transition from Meta I to Meta II, our data reveal a partial deprotonation of the retinal Schiff base preceding the conformational change of the receptor protein to Meta II. The technique and the results are discussed in regard to its advantages as well as its limitations.
authors.affiliation: Arbeitsgruppe Biophysik, Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 9, D-79104 Freiburg, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Lüdeke
authors.forename: Víctor A
authors.initials: VA
authors.lastname: Lórenz Fonfría
authors.forename: Friedrich
authors.initials: F
authors.lastname: Siebert
authors.forename: Reiner
authors.initials: R
authors.lastname: Vogel
mesh_terms: D000818:Animals; D002417:Cattle; D007700:Kinetics; D010777:Photochemistry; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry; D012160:Retina / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013995:Time
chemical_list: D020033:Protein Isoforms; D012545:Schiff Bases; D012243:Rhodopsin