We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-Lphenylalanine (pN3CH2Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN3CH2Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN3Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN3CH2Phe to different protein environments to be measured. The photo-stability of pN3CH2Phe was also measured relative to the photoreactive UAA, pN3Phe.

Two novel 2′-deoxyadenosine (dA) analogues, Si2-dA-SCN and Si2-dA-SeCN, and two novel phenylalanine (Phe) analogues, Boc-Me-PheCH2SCN and Boc-Me-PheCH2SeCN, have been synthesized and the thiocyanate (SCN) and selenocyanate (SeCN) functional groups evaluated as vibrational reporters. The syntheses of Si2-dA-SCN and Si2-dA-SeCN were accomplished in three steps in 16% and 32% overall yields, respectively, and the syntheses of Boc-Me-PheCH2SCN and Boc-Me-PheCH2SeCN were completed in four steps in 8.9% and 2.3% overall yields, respectively. The SCN and SeCN stretch vibrational modes were shown to be sensitive to the local environment by frequency shifts and full-width half-maximum (fwhm) changes in response to tetrahydrofuran (THF) and THF/water solvent mixtures. The vibrational lifetimes of the Si2-dA-SeCN (237±12 ps) and Boc-Me-PheCH2SeCN (295±31 ps) in THF solution were determined by ultrafast infrared pump-probe spectroscopy to be 1.5 to 3 times longer than those for Si2-dA-SCN (140±6 ps) and Boc-Me-PheCH2SCN (102±4 ps). The longer lifetimes for the SeCN analogues were attributed to the better insulating effects of the heavier selenium atom compared to the sulfur atom. The solvent sensitivity and longer vibrational lifetimes compared to other vibrational reporters suggest that SCN and SeCN vibrational reporters are well suited to studying several dynamic processes including protein and nucleic acid hydration and conformational changes, however stability issues may require post-synthetic modification methods to incorporate these reporters into biomacromolecules.

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.