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

Abstract

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...

Simple method to introduce an ester infrared probe into proteins

“…), comparison between the Fourier transform infrared (FTIR) spectra of YGGCGG and YGGC * GG obtained in D 2 O also validates the successful incorporation of the ME group. This is because the YGGC * GG peptide, but not the YGGCGG peptide, gives rise to an IR band within the region of 1700–1750 cm −1 . As expected, this ester carbonyl stretching vibrational band is broad and peaked at about 1720 cm −1 , which is similar to that observed for a short peptide containing a D M residue, due to hydrogen‐bonding interactions with the solvent molecules …”

“…Recently, Pazos et al have shown that the ester carbonyl stretching vibration is a useful and convenient site‐specific IR probe of protein electrostatics because (1) its frequency is linearly correlated with the local electrostatic field, even when the carbonyl group is engaged in hydrogen‐bonding interactions; (2) its frequency is located in the spectral range of 1700–1800 cm −1 , wherein no intrinsic protein vibrations show up at neutral pH; and (3) it has a relatively high extension coefficient. In particular, Pazos et al have tested and demonstrated the utility of two ester‐containing non‐natural amino acids, l ‐aspartic acid 4‐methyl ester (hereafter referred to as D M ) and l ‐glutamic acid 5‐methyl ester (hereafter referred to as E M ), by incorporating them into various model peptide systems via solid‐phase peptide synthesis. In contrast, other non‐natural amino acid‐based IR probes, such as p ‐cyanophenylalanine and p ‐azido‐phenylalanine, have been incorporated into large proteins in vivo via amber codon suppression .…”

Site-Specific Infrared Probes of Proteins

“…In a second example, Pazos et al (126) further showed that the C=O stretching frequency of two esters (i.e., methyl propionate and methyl acetate) afford similar spectroscopic properties as that of acetophenone. However, because the C=O stretching vibration of esters is typically in the range of 1710–1750 cm −1 , which decreases its spectral overlap with the amide I band of proteins, it offers certain advantages.…”

Vibrational Stark Effects of Carbonyl Probes Applied to Reinterpret IR and Raman Data for Enzyme Inhibitors in Terms of Electric Fields at the Active Site

“…Furthermore, vibrational solvatochromism can be used to systematically vary the field experienced by the probe in fluid solution, and the resulting spectral shifts can be related to the absolute electric field through MD simulations. 3–5 The separately measured Stark tuning rate (by VSS) provides a bridge between these two approaches. Additional computational methods have also been developed to determine the environmental effects on vibrational frequencies.…”

“…16–19 The carbonyl (C=O) vibration, which is generally more intense than nitriles, has been shown to vary linearly with electrostatic field in both H-bonding and non-H-bonding environments. 3, 5, 7–8, 20 The main limitation of the carbonyl probe is that its frequency overlaps with the densely populated amide I region in many biological systems. Fortunately, this can often be overcome by careful selection of a reference sample that is nearly identical to the sample of interest as well as isotopic labeling, or by using Raman spectroscopy.…”