Time-resolved thermodynamic changes photoinduced in 5,12-trans-locked bacteriorhodopsin. Evidence that retinal isomerization is required for protein activation.
Structural volume changes upon excitation of isomerization-blocked 5,12-trans-locked bacteriorhodopsin (bR) (bacterio-opsin + 5-12-trans-locked retinal) were studied using photothermal methods. The very small prompt expansion detected using laser-induced optoacoustics (0.3 mL/mol of absorbed photons) is assigned to a charge reorganization in the chromophore protein pocket concomitant with the formation of the intermediate T5.12. The subsequent contraction associated with a 300 ns lifetime is assigned to protein movements required to reach the entire chromoprotein free energy minimum, after the 17 ps optical decay of T5.12. The volume changes comprise the entropy of medium rearrangement during T5.12 formation and decay. The slow changes detected in previous studies by atomic force microscopy might be explained by the slowing down of movements in films containing 5,12-trans-locked bR. Photothermal beam deflection data with the 5,12-trans-locked bR suspensions indicate no further changes in microseconds to hundreds of milliseconds. Thus, all the absorbed energy is either released to the solution as heat or used for entropy changes within the first 300 ns after the pulse, supporting the paradigm that isomerization is required for signal transduction in retinal proteins. Bacterio-opsin assembled with all-trans-retinal afforded (similar to data reported with wild-type bR) an expansion of 2.6 mL/mol (assigned to the production of KE) followed by a further expansion of 0.8 mL/mol (KE-->KL; KE, KL, early and late K's) involving no heat loss. For KL decay to L, a contraction of 6 mL/mol of phototransformed reconstituted all-trans bR was determined.
Losi
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Max-Planck-Institut für Strahlenchemie, Mülheim an der Ruhr, Germany.
Michler
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Gärtner
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Braslavsky
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D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000528:radiation effects*; D010777:Photochemistry; D012172:Retinaldehyde / Q000737:chemistry / Q000528:radiation effects; D013053:Spectrophotometry; D013237:Stereoisomerism; D013816:Thermodynamics
D001436:Bacteriorhodopsins; D012172:Retinaldehyde