A Solvatochromic Model Calibrates Nitriles’ Vibrational Frequencies to Electrostatic Fields

Abstract

Electrostatic interactions provide a primary connection between a protein’s three-dimensional structure and its function. Infrared (IR) probes are useful because vibrational frequencies of certain chemical groups, such as nitriles, are linearly sensitive to local electrostatic field, and can serve as a molecular electric field meter. IR spectroscopy has been used to study electrostatic changes or fluctuations in proteins, but measured peak frequencies have not been previously mapped to total electric fields, because of the absence of a field-frequency calibration and the complication of local chemical effects such as H-bonds. We report a solvatochromic model that provides a means to assess the H-bonding status of aromatic nitrile vibrational probes, and calibrates their vibrational frequencies to electrostatic field. The analysis involves correlations between the nitrile’s IR frequency and its 13C chemical shift, whose observation is facilitated by a robust method for introducing isotopes into aromatic nitriles. The method is tested on the model protein Ribonuclease S (RNase S) containing a labeled p-CN-Phe near the active site. Comparison of the measurements in RNase S against solvatochromic data gives an estimate of the average total electrostatic field at this location. The value determined agrees quantitatively with MD simulations, suggesting broader potential for the use of IR probes in the study of protein electrostatics.

IR Probes of Protein Microenvironments: Utility and Potential for Perturbation

“…[21][22][23][24][25][26][27][28] The CN and N 3 probes have the advantage of being strong chromophores; however, unlike CÀD labels, CN and N 3 substituents are extrinsic probes that may introduce interactions that are not present in the native protein, and sensitivity to these artificial interactions may desensitize them to their native protein environment. Indeed, previous studies have indicated that both probes are predominantly sensitive to hydrogen bonding, [5,[29][30][31][32][33][34] interactions introduced by the probes themselves. Moreover, as extrinsic probes they might perturb the protein, a possibility exacerbated by the size of the N 3 group and by the H-bonding propensity of both CN and N 3 groups.…”

Solvatochromism and the solvation structure of benzophenone

“…9 The results herein suggest that these effects are readily accommodated by augmenting experimental spectroscopy with electronic structure calculations, thereby affording a means to rationalize any deviation from purely electrostatic contributions. Coupled with Kubo-Anderson lineshape analysis this tool becomes even more versatile, facilitating exploration of the vibrational probe's solvation shell.…”

“…Thus, while the calculated shifts are not identical to the experimental shifts, the use of reference data establish a correlation can provide a quantitative conversion factor. While prior studies avoided hydrogen bonding solvents due to difficulties in interpretation, 9 these concerns may be mitigated when theoretical calculations are used to augment experimental investigations. As a final note, the scatter and deviation from a linear dependence of the solvatochromic shift on the Onsager reaction field may be attributed, in part, to the crude nature of the Onsager approximation.…”

“…In fact, other nitrile and nitrile-free probes including alcohols, azides, fluorides, nitrosyl, and carbonyl compounds have been investigated both experimentally 2,4,19,20 and theoretically. [21][22][23][24][25] Furthermore, the aforementioned studies have all utilized the Stark effect, with a few notable exceptions 9,10 in which the nitrile solvatochromic shift was calibrated against a standard series of solvents. The latter approach is ideal as it drastically simplifies the measurement technique and permits observations over a broad range of physiologically relevant conditions.…”

Ultrafast dynamics induced by the interaction of molecules with electromagnetic fields: Several quantum, semiclassical, and classical approaches

“…Previously used nitrile probes for proteins include CN-labelled phenylalanine 289–291 and the nitrile-containing IDD type inhibitor for human aldose reductase 278,292 . Benzonitrile (PhCN) is another potentially useful probe to determine the local electrostatic environment as it fulfills three important criteria: the -CN stretching mode at $\approx 2200$ cm – 1 (a) absorbs in a frequency range (i.e., between 1800 cm – 1 and 2800 cm – 1 ) in which proteins containing only naturally occurring amino acids have no vibrational spectral response (except for the -SH group in cysteine), (b) is to a good approximation a local mode (i.e., uncoupled from other framework modes), and (c) the dipole moment of PhCN is to a large extent that of the nitrile group itself.…”