Expanding the Utility of 4-Cyano-L-Phenylalanine as a Vibrational Reporter of Protein Environments

Abstract

We present a number of theoretical results concerning the properties of and differences amongst several red fluorescent proteins (RFPs). RFPs are an extraordinarily useful group of fluorescent proteins that fluoresce at red-shifted wavelengths compared to GFP, thereby extending the color palette of fluorescent proteins for use in applications like FRET and multi-channel imaging. This redder fluorescence is caused by the extension of the pi-conjugated chain through oxidation of a second backbone bond of either Phenylalanine or Isoleucine, depending on the particular RFP. Our work has focused on the RFPs mCherry, mPlum, and DsRed, which share a common chromophore. Since these RFPs share a chromophore, differences in their photophysical properties must be due to differences in their chromophore environments. We performed a series of 100 ns molecular dynamics simulations followed by ZINDO semiempirical quantum mechanical calculations in an effort to explore variables such as excitation wavelength, quantum yield, and photostability in these RFPs. The ZINDO calculations were performed on MD simulation frames at 1 ps resolution. This fine resolution allowed for the observation of rare conformational states that suggest reasons for the known differences in quantum yield amongst the proteins. Notably, the electric field contributions from every nonchromophore protein atom and ion, and the~14,000 simulation cell water molecules, were used as a perturbation in the ZINDO calculations, which allowed us to query the electrostatic environmental influence on the chromophore.

Transparent Window Vibrational Probes for the Characterization of Proteins With High Structural and Temporal Resolution

“…212 Fayer and coworkers incorporated (N 3 )Phe into Mb at a site proximal to the heme moiety and used 2D IR spectroscopy to investigate structural dynamics. 213 Brewer and co-workers incorporated (CN)Phe 110,214 or (N 3 )Phe 215,216 into sfGFP to probe local protein environments. Budisa and co-workers incorporated (CN)Phe at position 36 of horse heart cyt c to investigate the response of the CN probe to changes in the oxidation state of the heme cofactor.…”

Kinetic Isotope Effect Provides Insight into the Vibrational Relaxation Mechanism of Aromatic Molecules: Application to Cyano-phenylalanine

“…We chose Phe-CN as our model system because (1) its CN stretching vibration is a useful site-specific infrared (IR) probe of proteins, 20 (2) Fayer and co-workers have shown that the CN vibrational lifetime of benzonitrile is shorter than that of its naturally abundant 13 CN isotopomer, 21 and (3) it is relatively straightforward to synthesize the four CN isotopomers of Phe-CN, 22 wherein the mass of the C (N) atom is ether 12 (14) or 13 (15). An additional goal of this study is to identify which one of these CN variants has the longest vibrational lifetime, as in certain applications the vibrational lifetime determines the detection time window of the measurement.…”

Molecular quantum mechanical gradients within the polarizable embedding approach—Application to the internal vibrational Stark shift of acetophenone

“…Vibrational probes have proven to be particularly well suited to act as local and directional metrics of protein electric fields via the vibrational Stark effect, [9][10][11][12][13][14][15][16][17] which describes the susceptibility of a vibrational transition to an electric field. Often one uses "internal" or "external" to distinguish the source of the Stark effect, being either a surrounding environment or an externally applied field.…”