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 gaining 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. Furthermore, 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, its application to amyloid fibrils formed by Aβ(16-22) revealed that the interior of such β-sheet assemblies has an ε value of approximately 5.6.

Keywords: IR spectroscopy; carbonyl groups; hydrogen bonds; protein electrostatics; vibrational probes.

Figure 1

Normalized FTIR spectra of MA and MP obtained in different solvents, as indicated. The concentration of the solute in each case was 20 mM and normalization is based on the integrated area of the band obtained in hexane (i.e., the spectra collected in other solvents were scaled so that their integrated areas are equal to that obtained in hexane). For MA in hexane, the peak absorbance was measured to be 0.0715, which gives rise to a molar extinction coefficient of 650 M^-1 cm^-1.

Figure 2

Center frequencies of the carbonyl stretching vibrations of MA (circles) and MP (squares) versus calculated local electric field for different solvents (represented by the same colors as those used in Figure 1). The solid lines are the best fits of these data to the linear equations indicated in the figure.

Figure 3

Offset FTIR spectra in the ester C=O stretching region of D_M-P, MA, E_M-P, and MP in different solvents, as indicated. The peptide concentrations were 2 mM and, in each case, the spectrum of the model compound has been normalized with respect to that of the corresponding peptide.

Figure 4

Normalized stretching vibrational bands of the ester carbonyl of 15 mM Aβ-D_M obtained immediately after the sample was prepared, after 7 days of incubation in D₂O, and also in the form of a dried film (dried under a flow of nitrogen for 7 days), as indicated. The band obtained with the dry film can be decomposed into 3 Gaussians (grey dashed lines) with the center frequencies given in the text.