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-tra ns-locked bacteriorhodopsin (bR) (bacterio-opsin + 5-12-trans-locked retina l) 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 T-5.12. The subsequent c ontraction associated with a 300 ns lifetime is assigned to protein movemen ts required to reach the entire chromoprotein free energy minimum, after th e 17 ps optical decay of T-5.12, The volume changes comprise the entropy of medium rearrangement during T-5.12 formation and decay, The slow changes d etected in previous studies by atomic force microscopy might be explained b y the slowing down of movements in films containing 5,12-trans-locked bR, P hotothermal beam deflection data with the 5,12-trans-locked bR suspensions indicate no further changes in microseconds to hundreds of milliseconds. Th us, all the absorbed energy is either released to the solution as heat or u sed for entropy changes within the first 300 ns after the pulse, supporting the paradigm that isomerization is required for signal transduction in ret inal proteins, Bacterio-opsin assembled with all-transretinal afforded (sim ilar to data reported with wild-type bR) an expansion of 2.6 mL/mol (assign ed to the production of K-E) followed by a further expansion of 0.8 mL/mol (K-E --> K-L; K-E, K-L, early and late K's) involving no heat loss. For K-L decay to L, a contraction of 6 mL/mol of phototransformed reconstituted al l-trans bR was determined.