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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. [ABSTRACT FROM AUTHOR]

Hydration is a key determinant of the folding, dynamics, and function of proteins. In this study, temperature-dependent Fourier transform infrared (FTIR) spectroscopy combined with singular value decomposition (SVD) and global fitting were used to investigate both the interaction of water with α-helical proteins and the cooperative thermal unfolding of these proteins. This methodology has been applied to an isolated α-helix (Fs peptide) and to globular α-helical proteins including the helical subdomain and full-length villin headpiece (HP36 and HP67). The results suggest a unique IR signature for the interaction of water with the helical amide carbonyl groups of the peptide backbone. The IR spectra indicate a weakening of the net hydrogen bond strength of water to the backbone carbonyls with increasing temperature. This weakening of the backbone solvation occurs as a discrete transition near the maximum of the temperature-dependent hydrophobic effect, not a continuous change with increasing temperature. Possible molecular origins of this effect are discussed with respect to previous molecular dynamics simulations of the temperature-dependent solvation of the helix backbone.

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. [ABSTRACT FROM AUTHOR]

[super 13]C[double bond][super 18]O isotope-edited infrared spectroscopy is used to examine the ability to resolve protein folding dynamics at the level of a single residue. The results have shown that a single [super 13]C[double bond][super 18]O label can be detected in a residue protein and that the signal-to-noise is enough for probing both equilibrium thermal unfolding and time-resolved folding dynamics.

We report a new method, microfluidic flow-flash, for measuring protein reaction kinetics. The method couples a microscope imaging detection system with a microfluidic flow cell to reduce data acquisition times and sample consumption. This combination allows for the simultaneous collection of spectral and temporal information. The microfluidic flow cell design utilizes three-dimensional sheath flow to reduce sample dispersion and minimize sample consumption. The ability to alter the flow rates in the microfluidic flow cells allows a variety of time scales to be studied with submillisecond time resolution. The imaging detection system can be coupled with several spectroscopic probes including fluorescence and UV/ visible absorbance spectroscopy. Here, we utilize the microfluidic flow-flash method to probe the kinetics of CO recombination or [O.sub.2] binding to myogiobin after the laser-induced photolysis of CO from myogiobin by UV/visible absorbance spectral imaging.

The first two structurally, magnetically, and spectroscopically characterized dinuclear valence tautomerism (VT) complexes wherein the bridging ligand is the ancillary diimine and all three possible valence tautomeric forms and exist below 500K are reported. A combination of magnetic susceptibility, electronic absorption spectroscopy, and X-ray absorption spectroscopy (XAS) at the Co K-edge to probe the effects of environment on VT equilibria.

Equilibrium Fourier transform infrared (FTIR) and temperature-jump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 [micro]s at 60.2[degrees]C) and double phenylalanine mutant (3.01 [micro]s at 49.9[degrees]C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 [micro]s at 49.9[degrees]C, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 [micro]s compared with 6.97 [micro]s for the WT at 49.9[degrees]C. protein folding | Fourier transform IR | temperature jump | diffusion collision

Variable angle reflectance FTIR spectroscopy was used to investigate the optical properties of indium tin oxide (ITO) and fluorine-doped tin oxide (SFO) thin films in the near-IR spectral region. The reflectance, plasma frequency, and electronic scattering time of ITO thin films were found to be dependent on the sheet resistance.

The iron-histidine band [V(sub Fe)-His] is assigned as a stretching mode in the deoxy ferrous state of histidine-ligated heme proteins. This involves a two-body motion involving the iron and histidine combined with a minor amount of heme doming.

Density functional theory (DFT) has been used to calculate the vibrational Stark tuning rates of a variety of nitriles and carbonyls in quantitative agreement with experimental values with a correction factor of f = 1.1 for the local electric field. These calculations show that the vibrational Stark tuning rate has an anharmonic contribution and a contribution due to geometric distortions caused in the molecules due to the applied electric field. The anharmonic and geometric distortion components of the vibrational Stark tuning rate were calculated by the frequency dependence of the CN or CO stretching mode with varying applied electric fields by using the optimized structure in zero applied field or allowing the structure to optimize in the applied electric field, respectively. The changes in the calculated frequency of the CN or CO stretching mode, bond length, and dipole moment of this bond with varying applied electric fields are shown. The transition polarizability and the difference polarizability were also calculated by DFT for comparison to the experimental data on nitriles and carbonyls. The DFT calculations suggest that the sign of the transition polarizability is negative and this result in turn has an effect on the experimental data analysis since the sign of the transition polarizability is not determined by experiment. [ABSTRACT FROM AUTHOR]

The recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9 is demonstrated. The analysis of the mutant proteins and model peptides has shown that the reduced native state fluorescence is due to the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains.

The selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that could function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding is reported. The methodology for the study of protein conformational changes using FRET could find general application to study the protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.

The recombination dynamics of NO with dehaloperoxidase (DHP) from Amphitrite ornata following photolysis are measured by femtosecond time-resolved absorption spectroscopy. The analysis of ligand recombination in photolyzed DHP-NO and H64V Mb-NO has provided evidence for the vital role of the distal histidine in stabilizing the NO ligand in the docking site, which provides a barrier to recombination that is modulated by protein dynamics and temperature.

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 - 3.5x10-2 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. [ABSTRACT FROM AUTHOR]

The Fourier transform infrared difference spectroscopy in conjunction with the photolytic release of caged nucleotides is used to identify and compare the specific structural changes induced by the binding of the two nucleotides, namely adenosine triphosphate (ATP) and adenosine diphosphate (ADP). It is found that the binding of ATP to RecA induces a conformation with a high affinity for DNA, whereas, ADP reveals a lower affinity for DNA with it.