The photophysical properties of the β-barrel superfolder green fluorescent protein (sfGFP) arise from the chromophore that forms post-translationally in the interior of the protein. Specifically, the protonation state of the side chain of tyrosine 66 in the chromophore, in addition to the network of hydrogen bonds between the chromophore and surrounding residues, is directly related to the electronic absorbance and emission properties of the protein. The pH dependence of the photophysical properties of this protein were modulated by the genetic, site-specific incorporation of 3-nitro-l-tyrosine (mNO2Y) at site 66 in sfGFP. The altered photophysical properties of this noncanonical amino acid (ncAA) sfGFP construct were assessed by absorbance and fluorescence spectroscopies. Notably, a comparison of the pK a of the 3-nitrophenol side chain of mNO2Y incorporated in the protein relative to the phenol side chain of the tyrosine at site 66 in the native chromophore as well as the pK a of the 3-nitrophenol side chain of the free ncAA were measured and are compared. A structural analysis of the ncAA containing sfGFP construct is presented to yield molecular insight into the origin of the altered absorbance and fluorescence properties of the protein.

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 N-3 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 (N-3) 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.

Unnatural amino acids (UAAs) have become useful tools for understanding protein structure and local environments. One of the most commonly used vibrational reporter UAAs to study local protein hydration environments is 4-cyano-L-phenylalanine (pCNF) due to the position and sensitivity of the nitrile symmetric stretching frequency as well as the ability to efficiently incorporate this UAA site-specifically into proteins using the Amber codon suppression methodology. Additionally, pCNF provides promise to function as a distance reporter when coupled with two-dimensional infrared (2D IR) spectroscopy. Here, a number of superfolder green fluorescent protein (sfGFP) constructs have been generated with pCNF site-specifically incorporated at two unique sites simultaneously. The pairs of sites were selected to permit the nitrile symmetric stretching frequency of pCNF at each site to be spectrally resolved. The temperature dependent linear IR spectra of these sfGFP constructs with two pCNF UAAs incorporated will be presented, illustrating that the nitrile groups are in different local environments. Preliminary X-ray crystal structures of these sfGFP constructs will be presented to probe if the UAAs resulted in a structural perturbation and to determine the distance between the nitrile groups of the two pCNF UAAs in each structure. These results will serve as the basis for and calibration of employing pCNF in future 2D IR spectroscopic studies to extract the distances between these two groups based upon coupling between them.

Unnatural amino acids (UAAs) containing vibrational reporters, such as nitrile, azide, and nitro groups, can be utilized to effectively probe local protein environments. Specifically, the nitrile symmetric stretching frequency of 4-cyano-L-phenylalanine (pCNF) is sensitive to local protein hydration and occurs in a relatively clear region of the infrared spectrum. In addition, the magnitude of change in this vibrational frequency over a given temperature range is dependent on the local protein environment of the nitrile group of pCNF. Finally, the UAA pCNF can also be site-specifically incorporated into proteins with high efficiency and fidelity using the Amber codon suppression methodology. Here, pCNF was incorporated into several different local protein environments in the enzyme Adenylate Kinase (AK). The successful incorporation was verified by time-of-flight mass spectrometry. These results in addition to the linear temperature-dependent IR analysis of these constructs to extract the hydration state of the nitrile group of pCNF in each of these AK constructs will be presented.

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.

Unnatural amino acids (UAAs) expand the landscape of protein biochemistry, providing new tools to examine and tune protein structure and function. Using the amber codon suppression technology these UAAs can be site-specifically incorporated into proteins. The UAA 4-cyano-L-phenylalanine (pCNF) contains the nitrile functional group whose nitrile stretching frequency is located both in a quiet region of protein IR spectra and is sensitive to local environment. Our work using pCNF site-specifically incorporated at various locations in both the gas-sensing Heme Nitric Oxide and/or Oxygen (H-NOX) binding protein from Caldanaerobacter subterraneus and the super folder Green Fluorescent Protein (sfGFP) will be discussed.

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 β-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.

An increasingly useful method of studying local electrostatic environments within protein molecules is through the incorporation of unnatural amino acids (UAAs) containing distinguishable chemical bonds with specific features in the infrared spectrum (IR) that change based on local environments. Of the existing UAAs, 4-cyano-L-phenylalanine (pCNF) is arguably one of the most useful vibrational reporters due to the fact that it is easily and affordably synthesized, it is well-studied, and its nitrile stretch appears in a biologically quiet region of the IR spectrum. In this study, pCNF has been genetically incorporated individually into three distinct sites of superfolder green fluorescent protein (sfGFP) via the amber codon suppression methodology. According to the wild type crystal structure of sfGFP, each of the selected sites are expected to have a unique solvation environment. Temperature-dependent infrared (IR) spectroscopy was utilized in order to test assess the local solvation environments of each of these sites. By measuring the nitrile stretching frequency as a function of temperature, the degree to which the incorporated pCNF participates in hydrogen bonding was determined. IR data and X-ray crystal structures from these constructs will be presented.

The unnatural amino acid 4-cyano-L-phenylalanine (pCNF) can be used as an infrared (IR) spectroscopic reporter to probe local chemical environments within the context of a larger protein. In this work we use molecular dynamics (MD) simulations to understand the structural basis for the IR spectrum observed when pCNF is incorporated into a particular site of super-folder green fluorescent protein (sfGFP). The observed nitrile IR absorbance band for pCNF at this site contains three subcomponents, which is in stark contrast to when pCNF is incorporated at other sites and only a single spectroscopic component is observed. Our simulation results suggest that these three subcomponents may be due to specific through-space contacts between the cyano group of pCNF and other side chains in the immediate vicinity of pCNF. We then performed MD simulations of single and double mutants of the protein - with pCNF in the same location - to predict which contacts with the cyano group will change and therefore which subcomponents of the IR spectrum may change. These results serve to motivate further experimental study of this system to help us better understand the overall effectiveness of pCNF as a spectroscopic reporter and the particular interactions occurring near our site of interest.

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.

Spectroscopic reporter unnatural amino acids (UAAs) have the potential to serve as effective, site-specific probes of local protein structure and dynamics. Specifically, the vibrational reporter UAA 4-cyano-L-phenylalanine (pCNF) is an effective probe of local protein environments due to the position of the nitrile symmetric stretch, the sensitivity of this stretch, and its small size. Here, pCNF was genetically incorporated individually into three distinct sites of superfolder green fluorescent protein (sfGFP) via the amber codon suppression methodology. Temperature-dependent infrared (IR) spectroscopy was utilized to assess the local solvation environments of each of these sites. One site was found to be fully solvated while a second site was partially solvated. The third site resulted in an IR spectrum whose nitrile IR absorbance band consisted of three subcomponents unlike the other two sites whose nitrile IR absorbance band consisted of a single component. Furthermore, each of the three subcomponents had a unique temperature dependence of the nitrile stretching frequency. Molecular dynamics (MD) simulations, in conjunction with X-ray crystallography, were utilized to identify specific protein-protein or protein-solvent interactions potentially responsible for the three components. Based upon this analysis, mutagenesis was performed surrounding the incorporated UAA. Subsequent temperature-dependent IR characterization of these constructs aided in the determination of the possible molecular origin of the multiple components present in the nitrile IR absorbance band. Data resulting from these constructs will be presented.

Unnatural amino acids (UAAs) consisting of a vibrational reporter such as a nitrile, azide, or nitro group have the potential to serve as effective, site-specific probes of local protein environments. Specifically the UAA 4-cyano-L-phenylalanine (pCNF) contains a nitrile vibrational reporter that is an effective probe of local environment due to the position and sensitivity of the nitrile symmetric stretch to local environment, in addition to the relative small size of the nitrile group. Additionally, this UAA can be site-specifically incorporated into proteins using the amber codon suppression method. Here, this vibrational reporter UAA was genetically incorporated at multiple distinct sites in the enzyme adenylate kinase (AK), which catalyzes the conversion of two adenylate diphosphate (ADP) molecules to one adenosine monophosphate (AMP) molecule and one adenosine triphosphate (ATP) molecule. Multiple sites were selected to represent a myriad of local solvation states some of which are predicted to change upon nucleotide binding based upon the X-ray crystal structures of apo and nucleotide bound AK. These local environments will be assessed with temperature-dependent infrared (IR) spectroscopy utilizing the correlation of the temperature-dependence of the nitrile stretching frequency of pCNF to local environment. Here the expression, purification and mass spectral characterization of these AK constructs containing pCNF will be presented.

Superfolder green fluorescent protein (sfGFP) contains an internal chromophore consisting of residues Thr65-Tyr66-Gly67. The protonation state of Tyr66 is directly correlated with the photophysical properties of the protein. Thus, a series of modified tyrosine unnatural amino acids (UAAs) were used to alter the photophysical properties of sfGFP. Specifically, 3-nitro-L-tyrosine (mNO2Y), 3-chloro-L-tyrosine (mClY), 3-bromo-L-tyrosine (mBrY), 3-iodo-L-tyrosine (mIY), 3-nitro-L-phenylalanine (pNO2F) and 3-cyano-L-phenylalanine (pCNF) were site-specifically incorporated at the 66 site in sfGFP using the amber codon suppression methodology. Prior to incorporation at the 66 site, optimization of the UAA incorporation into sfGFP was performed using a solvated site (133) and a partially buried site (149) in the protein since these sites do not alter the chromophore of the protein. The subsequent protein constructs were characterized by UV-Vis spectroscopy and fluorescence spectroscopy. The expression, purification, and spectral characterization of these protein constructs will be presented.

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.

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.

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.

The active sites of proteases were probed utilizing inhibitors modified with vibrational reporters and protein X-ray crystallography. Specifically, the inhibitors 1-cyano-benzene-4-sulfonylfluoride (CBSF) and 1-azido-benzene-4-sulfonylfluoride (ABSF) were synthesized containing the nitrile and azide vibrational reporters, respectively and reacted with the serine proteases subtilisin and trypsin to probe the active sites of the enzymes. Protein crystal structures of a number of these novel protein-inhibitor complexes have been determined using X-ray crystallography to illustrate successful incorporation of these inhibitors exclusively into the active site of these enzymes. This work shows that the tertiary structure of the active site closely matches that of previously published inhibitor-bound protease structures. Details about the X-ray crystal structures of the various protein-inhibitor complexes will be discussed. Complementary infrared spectroscopic results will also be presented to illustrate the ability of this combined approach to effectively probe the active site of proteases with inhibitors containing vibrational reporters.