Measuring Electrostatic Fields in Both Hydrogen-Bonding and Non-Hydrogen-Bonding Environments Using Carbonyl Vibrational Probes

Abstract

Vibrational probes can provide a direct read-out of the local electrostatic field in complex molecular environments, such as protein binding sites and enzyme active sites. This information provides an experimental method to explore the underlying physical causes of important biomolecular processes such as binding and catalysis. However, specific chemical interactions such as hydrogen bonds can have complicated effects on vibrational probes and confound simple electrostatic interpretations of their frequency shifts. We employ vibrational Stark spectroscopy along with infrared spectroscopy of carbonyl probes in different solvent environments and in Ribonuclease S to understand the sensitivity of carbonyl frequencies to electrostatic fields, including those due to hydrogen bonds. Additionally, we carried out molecular dynamics simulations to calculate ensemble-averaged electric fields in solvents and in Ribonuclease S, and found excellent correlation between calculated fields and vibrational frequencies. These data enabled the construction of a robust field-frequency calibration curve for the C=O vibration. The present results suggest that carbonyl probes are capable of quantitatively assessing the electrostatics of hydrogen bonding, making them promising for future study of protein function.

Molecular quantum mechanical gradients within the polarizable embedding approach—Application to the internal vibrational Stark shift of acetophenone

“…Thus, in contrast to Ref. 30, we find no evidence for H-bonding with chloroform from the present MD runs. The H-bonding in water can explain why the variation in C==O frequency is much larger in water than in the other solvents.…”

“…Acetophenone is a model compound for the unnatural amino acid p-acetylphenylalanine, which can be site-specifically introduced into proteins by several methods. 29 The carbonyl probe has attracted increasing attention due to the observations that (i) it has a rather large oscillator strength and Stark tuning rate, 30 (ii) its frequency varies linearly with the field also for hydrogen bonding solvents, as opposed to nitrile vibrations, 31 thus making it applicable across both hydrogen and non-hydrogen bonding environments, and (iii) its experimental use in proteins has been demonstrated by overcoming the complication related to the C==O spectral overlap with the intrinsic amide I vibration of the peptide bonds 32 by a suitable choice of reference sample. 30,33 Here, we perform classical MD simulations of the acetophenone-solvent systems, and the PE-DFT model is used to compute the C==O vibrational frequency as well as the corresponding local electric field sensed by acetophenone in the geometrically frozen solvent cages for each MD configuration.…”

Ester Carbonyl Vibration as a Sensitive Probe of Protein Local Electric Field

“…For example, upon changing the solvent from hexane to DMSO, the center frequency of MA is red-shifted by 18.1 cm -1 , compared to the 14.4 cm -1 shift observed for p-Ac-Phe. [7] Thus, these results substantiate the utility of these ester carbonyl stretching vibrations as sensitive probes of the local electric field, provided that a quantitative relationship between the electric field and frequency can be determined.Interestingly, while in aprotic solvents the ester carbonyl stretching vibration results in a single absorption band, in protic solvents, where H-bonding between the vibrator and solvent is possible, the linear IR spectra contain more than one resolvable feature, suggesting that differently solvated or H-bonded species are present. Such spectral features have also been observed for nitrile and amide modes in protic solvents such as methanol.…”

“…For example, upon changing the solvent from hexane to DMSO, the center frequency of MA is red-shifted by 18.1 cm -1 , compared to the 14.4 cm -1 shift observed for p-Ac-Phe. [7] Thus, these results substantiate the utility of these ester carbonyl stretching vibrations as sensitive probes of the local electric field, provided that a quantitative relationship between the electric field and frequency can be determined.…”

“…The computational study of Cho and coworkers has predicted that the stretching mode of such a carbonyl is not only localized, but its frequency also varies linearly with the electrostatic field for both H-bonding and non-H-bonding environments, [6] thus making it an ideal candidate for the aforementioned applications. Indeed, Boxer and coworkers [7] have recently shown that the C=O stretching frequency of p-acetyl-L-phenylalanine (p-Ac-Phe) can serve as a reporter of the local electrostatic field of proteins. However, this vibrational transition (at ~1673 cm -1 ) overlaps with the protein amide I band and, thus its application requires careful background subtraction using the wild-type protein.…”