Unusual Spectroscopic and Electric Field Sensitivity of Chromophores with Short Hydrogen Bonds: GFP and PYP as Model Systems

Abstract

Short hydrogen bonds, with heavy-atom distances less than 2.7 Å, are believed to exhibit proton delocalization and their possible role in catalysis has been widely debated. While spectroscopic and/or structural methods are usually employed to study the degree of proton delocalization, ambiguities still arise and no direct information on the corresponding potential energy surface is obtained. Here we apply an external electric field to perturb the short hydrogen bond(s) within a collection of green fluorescent protein S65T/H148D variants and photoactive yellow protein mutants, where the chromophore participates in the short hydrogen bond(s) and serves as an optical probe of the proton position. As the proton is charged, its position may shift in response to the external electric field, and the chromophore's electronic absorption can thus reflect the ease of proton transfer. The results suggest that low-barrier hydrogen bonds are not present within these proteins even when proton affinities between donor and acceptor are closely matched. Exploiting the chromophores as pre-calibrated electrostatic probes, the covalency of short hydrogen bonds as a non-electrostatic component was also revealed. No clear evidence was found for a possible contribution of unusually large polarizabilities of short hydrogen bonds due to proton delocalization; a theoretical framework for this interesting phenomenon is developed.

TDDFT Investigation of the Raman and Resonant Raman Spectra of Fluorescent Protein Chromophore Models

“…This issue has only scarcely been investigated experimentally. Raman spectra of GFP-like proteins in a presumed pure neutral cis ( Z ) form have been reported in several publications, with varying degrees of correspondence between spectra of the model chromophore and the full protein. ,,,,− In most of these studies, there is a good correspondence between the spectra of the model and the protein in the high-wavenumber region (three characteristic peaks between 1550 and 1650 cm –1 ), but not necessarily in the lower-wavenumber region. For example, the spectrum of the bright form of dronpa, reported by Higashino and recorded at resonant conditions, shows peaks at 1090, 1172, and 1366 cm –1 , but the model compound has its most significant peaks in this region at 1028, 1173, 1228, and 1290 cm –1 .…”

Photoactive Yellow Protein Represents a Distinct, Evolutionarily Novel Family of PAS Domains

“…This suggests that besides Cys69, at least one of the other two residues is required for hydrogen bond formation and retaining the photocycle. A study on S. ruber showed that in the PYP Y42F/E46Q double mutant, pCA always prefers the protonated state ( 70 ), which may cause photocycle initiation to fail.…”

Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme

“…It is the ability to prepare subtle mutants or variants that facilitates deeper mechanistic studies and energetic probing, in combination with rigorous characterizations, such as spectroscopic, kinetic, functional, and structural analyses and theoretical modeling. Subtle electrostatic/electronic and steric modulation to critical structural elements and/or physical properties for protein functions, including but not limited to cation−π interactions, 67 π−π interactions, 23 hydrogen bonds, 68,69 dipole−π interactions, 63 pK a , 70 nucleophilicity, 71 electronic distribution, 22 redox potential, 72 active-site electric field, 73 and isomerization efficiency, 74 can then be quantified by a suitable parameter that is derived from the physical chemistry or physical organic 43 tradition. The explored sequence space can accordingly be encoded with such parameter to interrogate its importance on energetics during catalysis, providing insights into the design principles of the large classes of enzymes adopting polar or radical mechanisms.…”