Inhibition of vibrational energy flow within an aromatic scaffold via heavy atom effect.

The regulation of intramolecular vibrational energy redistribution (IVR) to influence energy flow within molecular scaffolds provides a way to steer fundamental processes of chemistry, such as chemical reactivity in proteins and design of molecular diodes. Using two-dimensional infrared (2D IR) spectroscopy, changes in the intensity of vibrational cross-peaks are often used to evaluate different energy transfer pathways present in small molecules. Previous 2D IR studies of para-azidobenzonitrile (PAB) demonstrated that several possible energy pathways from the N3 to the cyano-vibrational reporters were modulated by Fermi resonance, followed by energy relaxation into the solvent [Schmitz et al., J. Phys. Chem. A 123, 10571 (2019)]. In this work, the mechanisms of IVR were hindered via the introduction of a heavy atom, selenium, into the molecular scaffold. This effectively eliminated the energy transfer pathway and resulted in the dissipation of the energy into the bath and direct dipole-dipole coupling between the two vibrational reporters. Several structural variations of the aforementioned molecular scaffold were employed to assess how each interrupted the energy transfer pathways, and the evolution of 2D IR cross-peaks was measured to assess the changes in the energy flow. By eliminating the energy transfer pathways through isolation of specific vibrational transitions, through-space vibrational coupling between an azido (N3) and a selenocyanato (SeCN) probe is facilitated and observed for the first time. Thus, the rectification of this molecular circuitry is accomplished through the inhibition of energy flow using heavy atoms to suppress the anharmonic coupling and, instead, favor a vibrational coupling pathway.

Unraveling Complex Local Protein Environments with 4-Cyano-l-phenylalanine.

We present a multifaceted approach to effectively probe complex local protein environments utilizing the vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNPhe) in the model system superfolder green fluorescent protein (sfGFP). This approach combines temperature-dependent infrared (IR) spectroscopy, X-ray crystallography, and molecular dynamics (MD) simulations to provide a molecular interpretation of the local environment of the nitrile group in the protein. Specifically, a two-step enantioselective synthesis was developed that provided an 87% overall yield of pCNPhe in high purity without the need for chromatography. It was then genetically incorporated individually at three unique sites (74, 133, and 149) in sfGFP to probe these local protein environments. The incorporation of the UAA site-specifically in sfGFP utilized an engineered, orthogonal tRNA synthetase in E. coli using the Amber codon suppression protocol, and the resulting UAA-containing sfGFP constructs were then explored with this approach. This methodology was effectively utilized to further probe the local environments of two surface sites (sites 133 and 149) that we previously explored with room temperature IR spectroscopy and X-ray crystallography and a new interior site (site 74) featuring a complex local environment around the nitrile group of pCNPhe. Site 133 was found to be solvent-exposed, while site 149 was partially buried. Site 74 was found to consist of three distinct local environments around the nitrile group including nonspecific van der Waals interactions, hydrogen-bonding to a structural water, and hydrogen-bonding to a histidine side chain.

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein.

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the ß-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

Extending the vibrational lifetime of azides with heavy atoms.

The development of novel vibrational reporters (VRs), aka infrared (IR) probes, to study local environments and dynamic processes in biomolecules and materials continues to be an important area of research. Azides are important VRs because of their small size and large transition dipole strengths, however, their relatively short vibrational lifetimes (<2 ps) have limited their full potential. Herein we report that the vibrational lifetimes of azides can be increased by attaching them to heavy atoms and by using heavy 15N isotopes. Three group 14 atom triphenyl azides (Ph3CN3, Ph3SiN3, Ph3SnN3), and their triple-15N isotopomers, were synthesized in good yields. Tributyltin azide and its heavy isotopomer (Bu3Sn15N3) were also prepared to probe the effect of molecular scaffolding. The extinction coefficients for the natural abundance azides were determined, ranging from 900 to 1500 M-1 cm-1. The vibrational lifetimes of all azides were measured by pump-probe IR spectroscopy and each showed a major component with a short-to-moderate vibrational lifetime and a minor component with a much longer vibrational lifetime. Based on these results, the lifetime, aka the observation window, of an azide reporter can be extended from ∼2 ps to as long as ∼300 ps by a combination of isotopic labeling and heavy atom effect. 2D IR measurements of these compounds further confirmed the ability to observe these azide transitions at much longer timescales showing their utility to capture dynamic processes from tens to hundreds of picoseconds.

2D-IR studies of cyanamides (NCN) as spectroscopic reporters of dynamics in biomolecules: Uncovering the origin of mysterious peaks.

Cyanamides (NCN) have been shown to have a larger transition dipole strength than cyano-probes. In addition, they have similar structural characteristics and vibrational lifetimes to the azido-group, suggesting their utility as infrared (IR) spectroscopic reporters for structural dynamics in biomolecules. To access the efficacy of NCN as an IR probe to capture the changes in the local environment, several model systems were evaluated via 2D IR spectroscopy. Previous work by Cho [G. Lee, D. Kossowska, J. Lim, S. Kim, H. Han, K. Kwak, and M. Cho, J. Phys. Chem. B 122(14), 4035-4044 (2018)] showed that phenylalanine analogues containing NCN show strong anharmonic coupling that can complicate the interpretation of structural dynamics. However, when NCN is embedded in 5-membered ring scaffolds, as in N-cyanomaleimide and N-cyanosuccinimide, a unique band structure is observed in the 2D IR spectrum that is not predicted by simple anharmonic frequency calculations. Further investigation indicated that electron delocalization plays a role in the origins of the band structure. In particular, the origin of the lower frequency transitions is likely a result of direct interaction with the solvent.

Tuning Molecular Vibrational Energy Flow within an Aromatic Scaffold via Anharmonic Coupling.

From guiding chemical reactivity in synthesis or protein folding to the design of energy diodes, intramolecular vibrational energy redistribution harnesses the power to influence the underlying fundamental principles of chemistry. To evaluate the ability to steer these processes, the mechanism and time scales of intramolecular vibrational energy redistribution through aromatic molecular scaffolds have been assessed by utilizing two-dimensional infrared (2D IR) spectroscopy. 2D IR cross peaks reveal energy relaxation through an aromatic scaffold from the azido- to the cyano-vibrational reporters in para-azidobenzonitrile (PAB) and para-(azidomethyl)benzonitrile (PAMB) prior to energy relaxation into the solvent. The rates of energy transfer are modulated by Fermi resonances, which are apparent by the coupling cross peaks identified within the 2D IR spectrum. Theoretical vibrational mode analysis allowed the determination of the origins of the energy flow, the transfer pathway, and a direct comparison of the associated transfer rates, which were in good agreement with the experimental results. Large variations in energy-transfer rates, approximately 1.9 ps for PAB and 23 ps for PAMB, illustrate the importance of strong anharmonic coupling, i.e., Fermi resonance, on the transfer pathways. In particular, vibrational energy rectification is altered by Fermi resonances of the cyano- and azido-modes allowing control of the propensity for energy flow.

Paired Spectroscopic and Crystallographic Studies of Proteases.

The active sites of subtilisin and trypsin have been studied by paired IR spectroscopic and X-ray crystallographic studies. The active site serines of the proteases were reacted with 4-cyanobenzenesulfonyl fluoride (CBSF), an inhibitor that contains a nitrile vibrational reporter. The nitrile stretch vibration of the water-soluble inhibitor model, potassium 4-cyanobenzenesulfonate (KCBSO), and the inhibitor were calibrated by IR solvent studies in H2O/DMSO and the frequency-temperature line-slope (FTLS) method in H2O and THF. The inhibitor complexes were examined by FTLS and the slopes of the best fit lines for subtilisin-CBS and trypsin-CBS in aqueous buffer were both measured to be -3.5×10-2 cm-1/°C. These slopes were intermediate in value between that of KCBSO in aqueous buffer and CBSF in THF, which suggests that the active-site nitriles in both proteases are mostly solvated. The X-ray crystal structures of the subtilisin-CBS and trypsin-CBS complexes were solved at 1.27 and 1.32 Å, respectively. The inhibitor was modelled in two conformations in subtilisin-CBS and in one conformation in the trypsin-CBS. The crystallographic data support the FTLS data that the active-site nitrile groups are mostly solvated and participate in hydrogen bonds with water molecules. The combination of IR spectroscopy utilizing vibrational reporters paired with X-ray crystallography provides a powerful approach to studying protein structure.

Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-L-phenylalanine.

The X-ray crystal structures of two superfolder green fluorescent protein (sfGFP) constructs containing a genetically incorporated spectroscopic reporter unnatural amino acid, 4-nitro-L-phenylalanine (pNO2F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO2F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO2F sfGFP construct crystallized with two molecules per asymmetric unit in space group P3221 and the crystal structure was refined to 2.05 Šresolution. Crystals of Asn149pNO2F sfGFP contained one molecule of sfGFP per asymmetric unit in space group P4122 and the structure was refined to 1.60 Šresolution. The alignment of Asp133pNO2F or Asn149pNO2F sfGFP with wild-type sfGFP resulted in small root-mean-square deviations, illustrating that these residues do not significantly alter the protein structure and supporting the use of pNO2F as an effective spectroscopic reporter of local protein structure and dynamics.

Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine.

The vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNF) was genetically incorporated individually at three sites (5, 36, and 78) in the heme protein Caldanaerobacter subterraneus H-NOX to probe local hydration environments. The UAA pCNF was incorporated site-specifically using an engineered, orthogonal tRNA synthetase in E. coli. The ability of all of the pCNF-containing H-NOX proteins to form the ferrous CO, NO, or O2 ligated and unligated states was confirmed with UV-Vis spectroscopy. The solvation state at each site of the three sites of pCNF incorporation was assessed using temperature-dependent infrared spectroscopy. Specifically, the frequency-temperature line slope (FTLS) method was utilized to show that the nitrile group at site 36 was fully solvated and the nitrile group at site 78 was de-solvated (buried) in the heme pocket. The nitrile group at site 5 was found to be partially solvated suggesting that the nitrile group was involved in moderate strength hydrogen bonds. These results were confirmed by the determination of the X-ray crystal structure of the H-NOX protein construct containing pCNF at site 5.

Primary Amebic Meningoencephalitis Associated With Rafting on an Artificial Whitewater River: Case Report and Environmental Investigation.

Background: Naegleria fowleri is a thermophilic ameba found in freshwater that causes primary amebic meningoencephalitis (PAM) when it enters the nose and migrates to the brain. Patient exposure to water containing the ameba typically occurs in warm freshwater lakes and ponds during recreational water activities. In June 2016, an 18-year-old woman died of PAM after traveling to North Carolina, where she participated in rafting on an artificial whitewater river. Methods: We conducted an epidemiologic and environmental investigation to determine the water exposure that led to the death of this patient. Results: The case patient's most probable water exposure occurred while rafting on an artificial whitewater river during which she was thrown out of the raft and submerged underwater. The approximately 11.5 million gallons of water in the whitewater facility were partially filtered, subjected to ultraviolet light treatment, and occasionally chlorinated. Heavy algal growth was noted. Eleven water-related samples were collected from the facility; all were positive for N. fowleri. Of 5 samples collected from the nearby natural river, 1 sediment sample was positive for N. fowleri. Conclusions: This investigation documents a novel exposure to an artificial whitewater river as the likely exposure causing PAM in this case. Conditions in the whitewater facility (warm, turbid water with little chlorine and heavy algal growth) rendered the water treatment ineffective and provided an ideal environment for N. fowleri to thrive. The combination of natural and engineered elements at the whitewater facility created a challenging environment to control the growth of N. fowleri.

Two-Dimensional Infrared Study of Vibrational Coupling between Azide and Nitrile Reporters in a RNA Nucleoside.

The vibrations in the azide, N3, asymmetric stretching region and nitrile, CN, symmetric stretching region of 2'-azido-5-cyano-2'-deoxyuridine (N3CNdU) are examined by two-dimensional infrared (2D IR) spectroscopy. At earlier waiting times, the 2D IR spectrum shows the presence of both vibrational transitions along the diagonal and off-diagonal cross peaks indicating vibrational coupling. The coupling strength is determined from the off-diagonal anharmonicity to be 66 cm(-1) for the intramolecular distance of ∼7.9 Å, based on a structural map generated for this model system. In addition, the frequency-frequency correlation decay is detected, monitoring the solvent dynamics around each individual probe position. Overall, these vibrational reporters can be utilized in tandem to simultaneously track global structural information and fast structural fluctuations.

Synthesis and Evaluation of the Sensitivity and Vibrational Lifetimes of Thiocyanate and Selenocyanate Infrared Reporters.

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.

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

Varying the reduced mass of an oscillator via isotopic substitution provides a convenient means to alter its vibrational frequency and hence has found wide applications. Herein, we show that this method can also help delineate the vibrational relaxation mechanism, using four isotopomers of the unnatural amino acid p-cyano-phenylalanine (Phe-CN) as models. In water, the nitrile stretching frequencies of these isotopomers, Phe-(12)C(14)N (1), Phe-(12)C(15)N (2), Phe-(13)C(14)N (3), and Phe-(13)C(15)N (4), are found to be equally separated by ∼27 cm(-1), whereas their vibrational lifetimes are determined to be 4.0 ± 0.2 (1), 2.2 ± 0.1 (2), 3.4 ± 0.2 (3), and 7.9 ± 0.5 ps (4), respectively. We find that an empirical relationship that considers the effective reduced mass of CN can accurately account for the observed frequency gaps, while the vibrational lifetime distribution, which suggests an intramolecular relaxation mechanism, can be rationalized by the order-specific density of states near the CN stretching frequency.

Probing the effectiveness of spectroscopic reporter unnatural amino acids: a structural study.

The X-ray crystal structures of superfolder green fluorescent protein (sfGFP) containing the spectroscopic reporter unnatural amino acids (UAAs) 4-cyano-L-phenylalanine (pCNF) or 4-ethynyl-L-phenylalanine (pCCF) at two unique sites in the protein have been determined. These UAAs were genetically incorporated into sfGFP in a solvent-exposed loop region and/or a partially buried site on the ß-barrel of the protein. The crystal structures containing the UAAs at these two sites permit the structural implications of UAA incorporation for the native protein structure to be assessed with high resolution and permit a direct correlation between the structure and spectroscopic data to be made. The structural implications were quantified by comparing the root-mean-square deviation (r.m.s.d.) between the crystal structure of wild-type sfGFP and the protein constructs containing either pCNF or pCCF in the local environment around the UAAs and in the overall protein structure. The results suggest that the selective placement of these spectroscopic reporter UAAs permits local protein environments to be studied in a relatively nonperturbative fashion with site-specificity.

Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins.

An unnatural amino acid, 4-(2-azidoethoxy)-L-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54% overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the azide asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites by using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three-atom linker between the azido group and the aromatic ring of AePhe. An X-ray crystal structure of sfGFP-149-AePhe was solved at 1.45 Å resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP-AePhe constructs efficiently undergo a bioorthogonal strain-promoted click cycloaddition with a dibenzocyclooctyne derivative.

Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids.

Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-L-phenylalanine (1) and 4-azidomethyl-L-phenylalanine (2), form a series of unnatural amino acids (UAAs) containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine. These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein environments. Three of the four azido-modified phenylalanine residues were successfully genetically incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity. SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP indicated that the azide groups were hydrated in the protein.

Legionnaires' Disease Outbreak at a Long-Term Care Facility Caused by a Cooling Tower Using an Automated Disinfection System--Ohio, 2013.

On July 9, 2013, an outbreak of Legionnaires' disease (LD) was identified at Long-Term Care Facility A in central Ohio. This article describes the investigation of the outbreak and identification of the outbreak source, a cooling tower using an automated biocide delivery system. In total, 39 outbreak LD cases were identified; among these, six patients died. Water samples from a cooling tower were positive for Legionella pneumophila serogroup 1, reactive to monoclonal antibody 2, with matching sequence type to a patient isolate. An electronic control system turned off cooling tower pumps during low-demand periods, preventing delivery of disinfectant by a timed-release system, and leading to amplification of Legionella in the cooling tower. Guidelines for tower maintenance should address optimal disinfection when using automated systems.

Sensitive, site-specific, and stable vibrational probe of local protein environments: 4-azidomethyl-L-phenylalanine.

We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-L-phenylalanine (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 photostability of pN3CH2Phe was also measured relative to the photoreactive UAA, pN3Phe.

Early turn formation and chain collapse drive fast folding of the major cold shock protein CspA of Escherichia coli.

The folding mechanism of the ß-sheet protein CspA, the major cold shock protein of Escherichia coli, was previously reported to be a concerted, two-state process. We have reexamined the folding of CspA using multiple spectroscopic probes of the equilibrium transition and laser-induced temperature jump (T-jump) to achieve better time resolution of the kinetics. Equilibrium temperature-dependent Fourier transform infrared (1634 cm(-1)) and tryptophan fluorescence measurements reveal probe-dependent thermal transitions with midpoints (T(m)) of 66 ± 1 and 61 ± 1 °C, respectively. Singular-value decomposition analysis with global fitting of the temperature-dependent infrared (IR) difference spectra reveals two spectral components with distinct melting transitions with different midpoints. T-jump relaxation measurements of CspA probed by IR and fluorescence spectroscopy show probe-dependent multiexponential kinetics characteristic of non-two-state folding. The frequency-dependent IR transients all show biphasic relaxation with average time constants of 50 ± 7 and 225 ± 25 µs at a T(f) of 77 °C and almost equal amplitudes. Similar biphasic kinetics are observed using Trp fluorescence of the wild-type protein and the Y42W and T68W mutants, with comparable lifetimes. All of these observations support a model for the folding of CspA through a compact intermediate state. The transient IR and fluorescence spectra are consistent with a diffuse intermediate having ß-turns and substantial ß-sheet structure. The loop ß3-ß4 structure is likely not folded in the intermediate state, allowing substantial solvent penetration into the barrel structure.

Expanding the utility of 4-cyano-L-phenylalanine as a vibrational reporter of protein environments.

The ability to genetically incorporate amino acids modified with spectroscopic reporters site-specifically into proteins with high efficiency and fidelity has greatly enhanced the ability to probe local protein structure and dynamics. Here, we have synthesized the unnatural amino acid (UAA), 4-cyano-L-phenylalanine (pCNPhe), containing the nitrile vibrational reporter and three isotopomers ((15)N, (13)C, (13)C(15)N) of this UAA to enhance the ability of pCNPhe to study local protein environments. Each pCNPhe isotopic variant was genetically incorporated in an efficient, site-specific manner into superfolder green fluorescent protein (sfGFP) in response to an amber codon with high fidelity utilizing an engineered, orthogonal aminoacyl-tRNA synthetase. The isotopomers of 4-cyano-L-phenylalanine permitted the nitrile symmetric stretch vibration of these UAAs to be unambiguously assigned utilizing the magnitude and direction of the isotopic shift of this vibration. The sensitivity of the nitrile symmetric stretching frequency of each isotopic variant to the local environment was measured by individually incorporating the probes into two distinct local environments of sfGFP. The UAAs were also utilized in concert to probe multiple local environments in sfGFP simultaneously to increase the utility of 4-cyano-L-phenylalanine.