Chemical control of excited-state reactivity of the anionic green fluorescent protein chromophore.

Controlling excited-state reactivity is a long-standing challenge in photochemistry, as a desired pathway may be inaccessible or compete with other unwanted channels. An important example is internal conversion of the anionic green fluorescent protein (GFP) chromophore where non-selective progress along two competing torsional modes (P: phenolate and I: imidazolinone) impairs and enables Z-to-E photoisomerization, respectively. Developing strategies to promote photoisomerization could drive new areas of applications of GFP-like proteins. Motivated by the charge-transfer dichotomy of the torsional modes, we explore chemical substitution on the P-ring of the chromophore as a way to control excited-state pathways and improve photoisomerization. As demonstrated by methoxylation, selective P-twisting appears difficult to achieve because the electron-donating potential effects of the substituents are counteracted by inertial effects that directly retard the motion. Conversely, these effects act in concert to promote I-twisting when introducing electron-withdrawing groups. Specifically, 2,3,5-trifluorination leads to both pathway selectivity and a more direct approach to the I-twisted intersection which, in turn, doubles the photoisomerization quantum yield. Our results suggest P-ring engineering as an effective approach to boost photoisomerization of the anionic GFP chromophore.

Simulation-guided engineering of split GFPs with efficient ß-strand photodissociation.

Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live-cell imaging. Split-GFPs are widely used to study protein-protein interactions by fusing proteins of interest to split GFP fragments that create a fluorophore upon typically irreversible complementation. Thus, controlled dissociation of the fragments is desirable. Although we have found that split strands can be photodissociated, the quantum efficiency of light-induced photodissociation of split GFPs is low. Traditional protein engineering approaches to increase efficiency, including extensive mutagenesis and screening, have proved difficult to implement. To reduce the search space, key states in the dissociation process are modeled by combining classical and enhanced sampling molecular dynamics with QM/MM calculations, enabling the rational design and engineering of split GFPs with up to 20-fold faster photodissociation rates using non-intuitive amino acid changes. This demonstrates the feasibility of modeling complex molecular processes using state-of-the-art computational methods, and the potential of integrating computational methods to increase the success rate in protein engineering projects.

Steric and Electronic Origins of Fluorescence in GFP and GFP-like Proteins.

Fluorescent proteins have become routine tools for biological imaging. However, their nanosecond lifetimes on the excited state present computational hurdles to a full understanding of these photoactive proteins. In this work, we simulate approximately 0.5 nanoseconds of ab initio molecular dynamics to elucidate steric and electronic features responsible for fluorescent protein behavior. Using green fluorescent protein (GFP) and Dronpa2─widely used fluorescent proteins with contrasting functionality─as case studies, we leverage previous findings in the gas phase and solution to explore the deactivation mechanisms available to these proteins. Starting with ground-state analyses, we identify steric (the distribution of empty pockets near the chromophore) and electronic (electric fields exerted on chromophore moieties) factors that offer potential avenues for rational design. Picosecond timescale simulations on the excited state reveal that the chromophore can access twisted structures in Dronpa2, while the chromophore is largely confined to planarity in GFP. We couple ab initio multiple spawning (AIMS) and enhanced sampling simulations to discover and characterize conical intersection seams that facilitate internal conversion, which is a rare event in both systems. Our AIMS simulations correctly capture the relative fluorescence profiles of GFP and Dronpa2 within the first few picoseconds, and we attribute the diminished fluorescence intensity of Dronpa2, relative to GFP, to flexible chromophore intermediates on the excited state. Furthermore, we predict that twisted chromophore intermediates produce red-shifted intensities in the Dronpa2 fluorescence spectrum. If confirmed experimentally, this spectroscopic signature would provide valuable insights when screening and developing novel fluorescent proteins.

Internal conversion of the anionic GFP chromophore: in and out of the I-twisted S1/S0 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 S1 trapping along the P-torsion. The majority of the population (∼70%) is transferred to S0 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.

Resolving the ultrafast dynamics of the anionic green fluorescent protein chromophore in water.

The chromophore of the green fluorescent protein (GFP) is critical for probing environmental influences on fluorescent protein behavior. Using the aqueous system as a bridge between the unconfined vacuum system and a constricting protein scaffold, we investigate the steric and electronic effects of the environment on the photodynamical behavior of the chromophore. Specifically, we apply ab initio multiple spawning to simulate five picoseconds of nonadiabatic dynamics after photoexcitation, resolving the excited-state pathways responsible for internal conversion in the aqueous chromophore. We identify an ultrafast pathway that proceeds through a short-lived (sub-picosecond) imidazolinone-twisted (I-twisted) species and a slower (several picoseconds) channel that proceeds through a long-lived phenolate-twisted (P-twisted) intermediate. The molecule navigates the non-equilibrium energy landscape via an aborted hula-twist-like motion toward the one-bond-flip dominated conical intersection seams, as opposed to following the pure one-bond-flip paths proposed by the excited-state equilibrium picture. We interpret our simulations in the context of time-resolved fluorescence experiments, which use short- and long-time components to describe the fluorescence decay of the aqueous GFP chromophore. Our results suggest that the longer time component is caused by an energetically uphill approach to the P-twisted intersection seam rather than an excited-state barrier to reach the twisted intramolecular charge-transfer species. Irrespective of the location of the nonadiabatic population events, the twisted intersection seams are inefficient at facilitating isomerization in aqueous solution. The disordered and homogeneous nature of the aqueous solvent environment facilitates non-selective stabilization with respect to I- and P-twisted species, offering an important foundation for understanding the consequences of selective stabilization in heterogeneous and rigid protein environments.