Please use this identifier to cite or link to this item: https://hdl.handle.net/20.500.12202/3281
Title: Chromophore/protein interaction in sensory rhodopsin -I (SR-I) and bacteriorhodopsin (BR) in Halobacterium halobium, and flagellar motor switch modulation by SR-I signal generation
Authors: McCain, Donald Andrew
Keywords: Biophysics.
Issue Date: 1989
Publisher: ProQuest Dissertations & Theses
Citation: Source: Dissertation Abstracts International, Volume: 50-11, Section: B, page: 4871.;Advisors: John L. Spudich.
Abstract: To test the nature of the electrostatic interactions occurring between retinal and the protein, a series of retinal analogues with incrementally shortened {dollar}\pi{dollar}-systems and alterations in the {dollar}\beta{dollar}-ionone ring region were introduced into the retinal pocket of both SR-opsin and BR-opsin. Wavelength shifts in absorption due to analogue interactions with the protein microenvironment demonstrate that the same overall electrostatic and steric properties of the retinal binding site exist in both proteins despite their different functions. The experimental data in combination with molecular orbital calculations, resulted in a new description of the protein charge distribution in SR, and a revision of the previously proposed model for BR. A series of ring desmethyl and acyclic analogues of all-trans retinal were synthesized in order to test the importance of interactions occurring between the protein and the {dollar}\beta{dollar}-ionone ring region. Measurement of the absorption spectra and photochemical properties of these analogue pigments indicate that the C-5 methyl is both sufficient and necessary for a large opsin shift in SR and BR. The C-1 and C-5 methyl groups, even when present on an incomplete ring, are sufficient to generate the native absorption values of SR and BR. Flash photolysis of the analogue pigments indicates that the thermal reisomerization steps in the SR and BR photocycles are significantly affected by these analogues. A retinal deficient mutant which contains the SR apoprotein was reconstituted with the ring desmethyl and acyclic analogues. Phototactic responses to reversal-inducing saturating stimuli were determined by computized motion analysis at 37 C and the SR photocycle kinetics measured by flash photolysis of whole cells at 37 C. These in vivo t{dollar}{lcub}1\over2{rcub}{dollar} values ranged from 640 ms for all-trans retinal to 5.0 s for the slowest analogue pigment. The analogues reconstitute both SR attractant and two photon repellent responses. Cells with t{dollar}{lcub}1\over2{rcub}{dollar} values approximately twice that of the native pigment show similar attractant responses, while cells with SR kinetics 3{dollar}\times{dollar}-6{dollar}\times{dollar} slower, show shifts in the response distributions to longer times.;Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system.;The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing). After a reversal, the switch returns to state N, passing through an intermediate state 1 (inactive), which produces a ca. 2-s period of low reversal frequency before the state N Poisson rate is restored.
URI: https://ezproxy.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9003807
https://hdl.handle.net/20.500.12202/3281
Appears in Collections:Albert Einstein College of Medicine: Doctoral Dissertations

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