Nitrile bonds as infrared probes of electrostatics in ribonuclease S

Abstract

Three different nitrile-containing amino acids, p-cyanophenylalanine, m-cyanophenylalanine, and S-cyanohomocysteine, have been introduced near the active site of the semisynthetic enzyme ribonuclease S (RNase S) to serve as probes of electrostatic fields. Vibrational Stark spectra, measured directly on the probe-modified proteins, confirm the predominance of the linear Stark tuning rate in describing the sensitivity of the nitrile stretch to external electric fields, a necessary property for interpreting observed frequency shifts as a quantitative measure of local electric fields that can be compared with simulations. The X-ray structures of these nitrile-modified RNase variants and enzymatic assays demonstrate minimal perturbation to the structure and function, respectively, by the probes and provide a context for understanding the influence of the environment on the nitrile stretching frequency. We examine the ability of simulation techniques to recapitulate the spectroscopic properties of these nitriles as a means to directly test a computational electrostatic model for proteins, specifically that in the ubiquitous Amber-99 force field. Although qualitative agreement between theory and experiment is observed for the largest shifts, substantial discrepancies are observed in some cases, highlighting the ongoing need for experimental metrics to inform the development of theoretical models of electrostatic fields in proteins.

Figure 1

Structural models of X-ray data for RNase variants. (A) Ribbon diagram for pCN-RNase with the S-peptide in blue and S-protein in green. (B-E) Expanded view of the region around each nitrile from four aligned structures, with S-peptide in a blue cartoon representation, a semi-transparent surface representation of neighboring residues colored grey for carbon, blue for nitrogen and red for oxygen, and stick representations of the nitrile-modified residues: pCN-RNase (B; carbons in green), SCN-RNase (C; cyan), and the two independently modeled structures from the asymmetric unit of mCN-RNase: (α) carbons in yellow (D) and (β) carbons in magenta (E).

Figure 2

Electron density maps for mCN-RNase. (A) and (B) correspond to the chain names for the two monomers in the asymmetric unit in the PDB file. The experimental electron density map represented with wire mesh (2F_obs-F_calc contoured at 1 sigma in blue mesh, 1F_obs-F_calc contoured at 3 sigma in red mesh) for a model with the mCN-Phe residue modeled as Phe, showing missing density for the nitrile (see text). The names α and β are given to the conformer with the nitrile nearest Met13 and Phe120, respectively. (Two foreground residues removed from view for clarity).

Figure 3

FT-IR spectra of nitrile-modified RNase S and S-peptide. Top: normalized spectra of nitrile-modified S-peptide, free (blue) and bound to S-protein to form the RNase S complex (black) in 50% (vol/vol) glycerol in water, pH 7, 298 K. Bottom: Absorption spectra of the same set of RNase S complexes as above, taken at 80 K in black (left axis), and Stark spectra scaled to an applied field of 1 MV/cm in red (right axis), see Table 3.

Figure 4

Temperature dependence of the IR absorption spectra of nitrile-modified RNases in 50% glycerol/water, self-buffered at pH 7. Colors correspond to temperature: red, orange, yellow, green, blue, black (mCN-RNase only) equal, respectively, 23, 10, −5, −25, −35, −100° C.

Figure 5

Comparison between measured and simulated frequency shifts and distributions for CN-modified RNase variants. (A) Measured shifts in the peak frequency for SCN-, mCN- and pCN-RNase at pH 4.5 and pH 8.0 vs. the peak of the distribution of frequencies from 3 ns of MD (orange circles) or 3 ns of REMD (black squares) calculated for these same six cases with a post-processing dielectric of 2 (see text); experimental and simulated shifts are calculated relative to the value for EtSCN, m-tolunitrile or p-tolunitrile in n-hexane respectively (see text). Numbering as follows (pH 4.5 and pH 8.0, respectively): SCN = 1, 2; pCN = 3, 4; mCN = 6, 5. Best-fit lines: slope = 0.76 or 0.72; R² = 0.77 or 0.57 for REMD and MD respectively. (B) Widths of calculated frequency distributions versus experimental FWHM. Best-fit lines: slope = 0.81 or 0.80; R² = 0.87 or 0.73 for REMD and MD respectively.

Figure 6

Measured absorbance spectra (black) compared with calculated distributions of the instantaneous frequency (blue) both normalized to unit area with 0.2 cm⁻¹ spacing between bins. (A-C) pH 4.5. (D-F) pH 8.0. The reference frequency for the simulations is based on, from left to right, p-tolunitrile, m-tolunitrile and EtSCN in n-hexane, and is shown by the dashed vertical line. A post-processing dielectric of 2 was employed (see text).