Internal conversion of the anionic GFP chromophore: in and out of the I-twisted S 1 /S 0 conical intersection seam.

The functional diversity of the green fluorescent protein (GFP) family is intimately connected to the interplay between competing photo-induced transformations of the chromophore motif, anionic p -hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI - ). Its ability to undergo Z / E -isomerization is of particular importance for super-resolution microscopy and emerging opportunities in optogenetics. Yet, key dynamical features of the underlying internal conversion process in the native HBDI - chromophore remain largely elusive. We investigate the intrinsic excited-state behavior of isolated HBDI - to resolve competing decay pathways and map out the factors governing efficiency and the stereochemical outcome of photoisomerization. Based on non-adiabatic dynamics simulations, we demonstrate that non-selective progress along the two bridge-torsional ( i.e. , phenolate, P, or imidazolinone, I) pathways accounts for the three decay constants reported experimentally, leading to competing ultrafast relaxation primarily along the I-twisted pathway and S 1 trapping along the P-torsion. The majority of the population (∼70%) is transferred to S 0 in the vicinity of two approximately enantiomeric minima on the I-twisted intersection seam (MECI-Is). Despite their sloped, reactant-biased topographies (suggesting low photoproduct yields), we find that decay through these intersections leads to products with a surprisingly high quantum yield of ∼30%. This demonstrates that E -isomer generation results at least in part from direct isomerization on the excited state. A photoisomerization committor analysis reveals a difference in intrinsic photoreactivity of the two MECI-Is and that the observed photoisomerization is the combined result of two effects: early, non-statistical dynamics around the less reactive intersection followed by later, near-statistical behavior around the more reactive MECI-I. Our work offers new insight into internal conversion of HBDI - that both establishes the intrinsic properties of the chromophore and enlightens principles for the design of chromophore derivatives and protein variants with improved photoswitching properties.