Simulation analysis of the retinal conformational equilibrium in dark-adapted bacteriorhodopsin

In dark-adapted bacteriorhodopsin (bR) the retinal moiety populates two con formers: all-trans and (13,15)cis. Here we examine factors influencing the thermodynamic equilibrium and conformational transition between the two for ms, using molecular mechanics and dynamics calculations. Adiabatic potentia l energy mapping indicates that whereas the twofold intrinsic torsional pot entials of the C13=C14 and C15=N16 double bonds favor a sequential torsiona l pathway, the protein environment favors a concerted, bicycle-pedal mechan ism. Which of these two pathways will actually occur in bR depends on the a s yet unknown relative weight of the intrinsic and environmental effects. T he free energy difference between the conformers was computed for wild-type and modified bR, using molecular dynamics simulation. In the wild-type pro tein the free energy of the (13,15)cis retinal form is calculated to be 1.1 kcal/mol lower than the all-trans retinal form, a value within similar to k(B)T of experiment. In contrast, in isolated retinal the free energy of th e all-trans state is calculated to be 2.1 kcal/mol lower than (13,15)cis. T he free energy differences are similar to the adiabatic potential energy di fferences in the various systems examined, consistent with an essentially e nthalpic origin. The stabilization of the (13,15)cis form in bR relative to the isolated retinal molecule is found to originate from improved protein- protein interactions. Removing internal water molecules near the Schiff bas e strongly stabilizes the (13,15)cis form, whereas a double mutation that r emoves negative charges in the retinal pocket (Asp(85) to Ala; Asp(212) to Ala) has the opposite effect.