The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to yielding a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, is a convenient and sensitive probe in this regard since its frequency correlates linearly with the local electrostatic field for both hydrogen-bonding and non-hydrogen-bonding environments. We expect that the resultant frequency-electric field map will find use in various applications. In addition, we show that, when situated in a non-hydrogen bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, applying it to amyloid fibrils formed by Aβ [16][17][18][19][20][21][22] reveals that the interior of such β-sheet assemblies has a ε of ~5.6.
Keywords
IR Probe; Protein Electrostatics; Hydrogen BondingElectrostatic interactions are ubiquitous in biological molecules and, in many cases, play a key role in molecular association and enzymatic reactions. [1] However, quantifying the local electric field or how it changes inside a protein, especially in a site-specific manner and/or as a function of time, still remains a challenging task. One promising method in this regard is vibrational Stark spectroscopy, [2] which capitalizes on the fact that vibrational transitions have an intrinsic dependence on local electrostatic environment and uses an infrared (IR) probe that has a well-defined, localized vibrational mode to sense local electric field amplitude through the response of the frequency. [3] For example, the vibrational Stark effect has been used to determine the local electric field at protein interfaces and to monitor protein conformational transitions and dynamics. [4] While the theoretical underpinning of this methodology is straightforward, in practice the application of vibrational Stark spectroscopy to biological systems is currently limited by the availability of suitable vibrational probes. Herein, we show, using linear and nonlinear IR measurements and molecular dynamics (MD) simulations, that the ester carbonyl vibration in two non-natural amino acids can be used to quantitatively and site-specifically probe the electric fields of proteins, including those arising from hydrogen-bond (H-bond) interactions.The utility of a vibrational probe to reliably and conveniently measure local electric fields in proteins is evaluated by how well it meets several criteria. First and foremost, its frequency must show a sensitive and quantifiable dependence on the local electric field. Also, a chemical or biological method must exist to incorporate the probe into a protein.Furthermore, it must minimally perturb the native chemical and structural environment of interest. Finally, its vibration must be a localized mode having a large cross-section and, ideally, be located in an uncongested region of the IR spectru...