Critical Evaluation of Polarizable and Nonpolarizable Force Fields for Proteins Using Experimentally Derived Nitrile Electric Fields.

Molecular dynamics (MD) simulations are frequently carried out for proteins to investigate the role of electrostatics in their biological function. The choice of force field (FF) can significantly alter the MD results, as the simulated local electrostatic interactions lack benchmarking in the absence of appropriate experimental methods. We recently reported that the transition dipole moment (TDM) of the popular nitrile vibrational probe varies linearly with the environmental electric field, overcoming well-known hydrogen bonding (H-bonding) issues for the nitrile frequency and, thus, enabling the unambiguous measurement of electric fields in proteins (J. Am. Chem. Soc. 2022, 144 (17), 7562-7567). Herein, we utilize this new strategy to enable comparisons of experimental and simulated electric fields in protein environments. Specifically, previously determined TDM electric fields exerted onto nitrile-containing o-cyanophenylalanine residues in photoactive yellow protein are compared with MD electric fields from the fixed-charge AMBER FF and the polarizable AMOEBA FF. We observe that the electric field distributions for H-bonding nitriles are substantially affected by the choice of FF. As such, AMBER underestimates electric fields for nitriles experiencing moderate field strengths; in contrast, AMOEBA robustly recapitulates the TDM electric fields. The FF dependence of the electric fields can be partly explained by the presence of additional negative charge density along the nitrile bond axis in AMOEBA, which is due to the inclusion of higher-order multipole parameters; this, in turn, begets more head-on nitrile H-bonds. We conclude by discussing the implications of the FF dependence for the simulation of nitriles and proteins in general.

Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein Environments.

Nitriles are widely used vibrational probes; however, the interpretation of their IR frequencies is complicated by hydrogen bonding (H-bonding) in protic environments. We report a new vibrational Stark effect (VSE) that correlates the electric field projected on the -C≡N bond to the transition dipole moment and, by extension, the nitrile peak area or integrated intensity. This linear VSE applies to both H-bonding and non-H-bonding interactions. It can therefore be generally applied to determine electric fields in all environments. Additionally, it allows for semiempirical extraction of the H-bonding contribution to the blueshift of the nitrile frequency. Nitriles were incorporated at H-bonding and non-H-bonding protein sites using amber suppression, and each nitrile variant was structurally characterized at high resolution. We exploited the combined information available from variations in frequency and integrated intensity and demonstrate that nitriles are a generally useful probe for electric fields.

Photosynthetic reaction center variants made via genetic code expansion show Tyr at M210 tunes the initial electron transfer mechanism.

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine (M210) close to an essential electron transfer component via its replacement with site-specific, genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and X-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild type (WT). Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via a ∼4-ps and a ∼20-ps population to produce the charge-separated state P+HA- in all variants. Global analysis indicates that in the ∼4-ps population, P+HA- forms through a two-step process, P*→ P+BA-→ P+HA-, while in the ∼20-ps population, it forms via a one-step P* → P+HA- superexchange mechanism. The percentage of the P* population that decays via the superexchange route varies from ∼25 to ∼45% among variants, while in WT, this percentage is ∼15%. Increases in the P* population that decays via superexchange correlate with increases in the free energy of the P+BA- intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an ∼110-meV increase in the free energy of P+BA- along with a dramatic diminution of the 1,030-nm transient absorption band indicative of P+BA- formation. Collectively, this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.

Genetic Code Expansion in Rhodobacter sphaeroides to Incorporate Noncanonical Amino Acids into Photosynthetic Reaction Centers.

Photosynthetic reaction centers (RCs) are the membrane proteins responsible for the initial charge separation steps central to photosynthesis. As a complex and spectroscopically complicated membrane protein, the RC (and other associated photosynthetic proteins) would benefit greatly from the insight offered by site-specifically encoded noncanonical amino acids in the form of probes and an increased chemical range in key amino acid analogues. Toward that goal, we developed a method to transfer amber codon suppression machinery developed for E. coli into the model bacterium needed to produce RCs, Rhodobacter sphaeroides. Plasmids were developed and optimized to incorporate 3-chlorotyrosine, 3-bromotyrosine, and 3-iodotyrosine into RCs. Multiple challenges involving yield and orthogonality were overcome to implement amber suppression in R. sphaeroides, providing insights into the hurdles that can be involved in host transfer of amber suppression systems from E. coli. In the process of verifying noncanonical amino acid incorporation, characterization of this membrane protein via mass spectrometry (which has been difficult previously) was substantially improved. Importantly, the ability to incorporate noncanonical amino acids in R. sphaeroides expands research capabilities in the photosynthetic field.