Alasdair F. Bell

Ground state isomerization of a model green fluorescent protein chromophore.

The relationship between ground state cis–trans isomerization and protonation state is explored for a model green fluorescent protein chromophore, 4‐hydroxybenzylidene‐1,2‐dimethylimidazolinone (HBDI). We find that the protonation state has only a modest effect on the free energy differences between cis and trans isomers and on the activation energies for isomerization. Specifically, the experimental free energy differences are 3.3, 8.8, and 9.6 kJ/mol for cationic, neutral, and anionic forms of HBDI, respectively, and the activation energies are 48.9, 54.8, and 54.8 kJ/mol for cationic, neutral, and anionic forms, respectively. Furthermore, these activation energies are much smaller than might be expected based on comparison with similar systems. These results suggest that there may be a sub‐population of the chromophore, which is nearly equally accessible to all three protonation states, through which thermal isomerization may proceed.

Stereoselectivity of Enoyl-CoA Hydratase Results from Preferential Activation of One of Two Bound Substrate Conformers

Enoyl-CoA hydratase catalyzes the hydration of trans-2-crotonyl-CoA to 3(S)- and 3(R)-hydroxybutyryl-CoA with a stereoselectivity (3(S)/3(R)) of 400,000 to 1. Importantly, Raman spectroscopy reveals that both the s-cis and s-trans conformers of the substrate analog hexadienoyl-CoA are bound to the enzyme, but that only the s-cis conformer is polarized. This selective polarization is an example of ground state strain, indicating the existence of catalytically relevant ground state destabilization arising from the selective complementarity of the enzyme toward the transition state rather than the ground state. Consequently, the stereoselectivity of the enzyme-catalyzed reaction results from the selective activation of one of two bound substrate conformers rather than from selective binding of a single conformer. These findings have important implications for inhibitor design and the role of ground state interactions in enzyme catalysis.

Gas-phase absorption properties of DsRed model chromophores

The absorption spectra of two compounds, RFP(1) and RFP(2), designed to model the chromophore of the Red Fluorescent Protein DsRed have been recorded in the gas phase with a heavy-ion storage ring technique. Both anions and cations were investigated. The electronic delocalization is greater in RFP(2) than in RFP(1) due to an additional CC double bond in conjugation with the π system, and the absorption bands of RFP(2) are red-shifted compared to those of RFP(1). Band maxima of the RFP(2) and RFP(1) anions are 549 nm and 521 nm, respectively, and of the cations 448 nm and 441 nm, respectively. These values are in good agreement with calculated HOMO–LUMO gaps at the B3LYP/6-311++G(2d,p)//PM3 level of theory: 559 nm and 496 nm for the RFP(2) and RFP(1) anions and 452 nm and 436 nm for the corresponding cations. The protein absorbs maximally at 558 nm and it is assumed that the chromophore is anionic. Hence, the electronic structure of the RFP(2) anion is close to that of the in vivo chromophore in its protein environment. A comparison is made between the two model chromophores and their well-known Green Fluorescent Protein homologue chromophore, as well as between different media (gas phase, solution phase). For the anionic gas-phase spectra, vibrational structures are clearly resolved for both compounds (hν0u2006=u2006382u2006±u200610 cm−1 for RFP(1) and 518u2006±u200610 cm−1 for RFP(2)) and are assigned to harmonic vibrational progressions due to collective motion of the entire chromophores. Based on calculations on model chromophores closer to the wild-type DsRed chromophore, we suggest that the protein environment forces the chromophore to adopt a planar geometry.

Ultrafast excited and ground-state isomerization dynamics of the Green Fluorescent Protein chromophore in solution

Ultrafast dispersed pump-dump-probe spectroscopy was applied to a model Green Fluorescent Protein chromophore in solution. Sub-ps photodynamics in the excited and ground State has been observed that is ascribed to a hula-twist isomerization mechanism.