Vibrational Probe Research Articles

Dynamics of Water Molecules and Ions in Concentrated Lithium Chloride Solutions Probed with Ultrafast 2D IR Spectroscopy.

Water and ion dynamics in concentrated LiCl solutions were studied using ultrafast 2D IR spectroscopy with the methyl thiocyanate (MeSCN) CN stretch as the vibrational probe. The IR absorption spectrum of MeSCN has two peaks, one peak for water associated with the nitrogen lone pair of MeSCN (W) and the other peak corresponding to Li+ associated with the lone pair (L). To extract the spectral diffusion (structural dynamics) of W and L species, we developed a method that isolates the peak of interest by subtracting the 2D Gaussian proxies of multiple interfering peaks. Center line slope data (normalized frequency-frequency correlation function) for 2D bands from the W and L are fit with triexponential functions. The fastest component (1.1-1.6 ps) is assigned to local hydrogen bond length fluctuations. The intermediate timescale (∼4.0 ps) corresponds to the hydrogen bond network rearrangement. The slowest component decays in ∼40 ps and corresponds to ion pair and ion cluster dynamics. The very similar W and L spectral diffusion indicates that the motions of the water and ions are strongly coupled. Orientational relaxations of the W and L species were extracted using a new method to eliminate the effects of overlapping peaks. The results show that MeSCN bound to water undergoes orientational relaxation significantly faster than MeSCN bound to Li+. The orientational and spectral diffusion results are compared. A Stark coupling model is used to extract the root mean square average electric field caused by the ion clouds along the CN moiety as a function of concentration.

Vibrational solvatochromism of the ester carbonyl vibration of PCBM in organic solutions

Ester carbonyl stretch in a widely used organic semiconducting material, [6,6]-phenyl-C61-butyric acid methyl ester (PCBM), has been utilized as a vibrational probe of molecular morphology in emerging electronic materials due to the sensitivity of its vibrational frequency to the local environment. Vibrational solvatochromic shift has been observed for dilute PCBM in organic solvents of varying polarity, but the spectral shift does not follow the order of solvent polarity, and its microscopic origin remains elusive. Here, we applied a mixed quantum/classical approach to simulate the infrared (IR) spectra for the ester carbonyl stretch of PCBM in dichloromethane, chloroform, and benzene. In this approach, the ester carbonyl group is treated quantum mechanically with a frequency map, and the rest of the system is described by molecular dynamics simulations. Based on the reasonable agreement with experimental IR spectra, we show that the specific directional interaction between the ester carbonyl group and its neighboring solvent molecules, which is not well captured by the solvent polarity, is primarily responsible for the observed solvatochromic shift. Furthermore, we find that the strength of this interaction also governs the solvation dynamics of the ester carbonyl group and the resulting frequency fluctuation, leading to a more inhomogeneously broadened spectrum for PCBM in chloroform compared to that in dichloromethane and benzene.

Re-orientation of water molecules in response to surface charge at surfactant interfaces.

We present a measurement of molecular orientation of water at charged surfactant aqueous interfaces as a function of surface charge density. The polarization dependent spectral line shapes of the water bend mode were measured by vibrational sum-frequency generation at the positively charged surfactant cetyltrimethylammonium bromide (CTAB)/water interface and negatively charged surfactant sodium dodecyl sulfate/water interface. Orientational analysis using the water bend mode as a vibrational probe, within the electric dipole approximation, reveals structural differences between these surfaces and quantifies how different hydrogen bonded species re-orient around the surfactant head groups as the surface charge density changes. As the concentration of the positively charged surfactant (CTAB) increases, the surface water molecules with free-OH groups reorient their hydrogen away from the bulk water and the C2v axis closer to the surface normal. This suggests that these free-OH molecules are in general located above the positively charged head groups of CTAB, and thus, the charge-dipole interaction pulls their oxygen "down" and pushes hydrogen "up." On the contrary, water molecules with two donor hydrogen bonds re-orient their hydrogen toward the bulk water, likely because most of these molecules are below the CTAB surfactant head groups.

Raman and Quantum Yield Studies of Trp48-d5 in Azurin: Closed-Shell and Neutral Radical Species.

Isotopologues are valuable vibrational probes that shift features in a vibrational spectrum while preserving the electronic structure of the molecule. We report the vibrational and electronic spectra of perdeuterated tryptophan in solution (l-Trp-d5), as Trp48-d5 in azurin, and as the photogenerated neutral tryptophan radical, Trp48-d5•, in azurin. The UV resonance Raman bands of the perdeuterated closed-shell tryptophan in solution and in azurin are lower in frequency relative to the protiated counterpart. The observed decrease in frequencies of l-Trp-d5 bands relative to l-Trp-h5 enables the analysis of vibrational markers of other amino acids, e.g., phenylalanine, that overlap with some modes of l-Trp-h5. The Raman intensities vary between l-Trp-d5 and l-Trp-h5; these differences likely reflect modifications in normal mode composition upon perdeuteration. Analysis of the W3, W6, and W17 modes suggests that the W3 mode retains its utility as a conformational marker; however, the H-bond markers W6 and W17 appear to be less sensitive upon perdeuteration. The neutral tryptophan radical, Trp48-d5•, was generated in azurin with a slightly lower radical quantum yield than for Trp48-h5•. The visible resonance Raman spectrum of Trp48-d5• is different from that of Trp48-h5•, especially in terms of relative intensities, and all assignable peaks decreased in frequency upon perdeuteration. The absorption and emission spectra of the perdeuterated closed-shell and radical species exhibited hypsochromic shifts of less than 1 nm relative to the protiated species. The data presented here indicate that l-Trp-d5 is a valuable probe of vibrational structure, with minimal modification of photoreactivity and photophysics compared to l-Trp-h5.

C-D Vibration at C2 Position of Imidazolium Cation as a Probe of the Ionic Liquid Microenvironment.

Unlike molecular solvents, imidazolium-based ionic liquids are entirely made of ions with spatial heterogeneity. There is a need for spectroscopic probes that can assess the microenvironment near the cations of these complex liquids. In this manuscript, we describe simple chemical procedures to label the C2 position of imidazolium cation with a C-D vibrational probe and show, through linear and nonlinear vibrational spectroscopies, that this C-D stretching mode can be a useful analytical tool to assess both the solvent microenvironment and solute-solvent interactions in imidazolium-based ionic liquids from the cation point of view. It is expected that this C-D vibration probe on the cation will lead to the development of innovative experimental strategies that can provide a better understanding of such ionic liquids.

Tracking Aqueous Proton Transfer by Two-Dimensional Infrared Spectroscopy and ab Initio Molecular Dynamics Simulations.

Proton transfer in water is ubiquitous and a critical elementary event that, via proton hopping between water molecules, enables protons to diffuse much faster than other ions. The problem of the anomalous nature of proton transport in water was first identified by Grotthuss over 200 years ago. In spite of a vast amount of modern research effort, there are still many unanswered questions about proton transport in water. An experimental determination of the proton hopping time has remained elusive due to its ultrafast nature and the lack of direct experimental observables. Here, we use two-dimensional infrared spectroscopy to extract the chemical exchange rates between hydronium and water in acid solutions using a vibrational probe, methyl thiocyanate. Ab initio molecular dynamics (AIMD) simulations demonstrate that the chemical exchange is dominated by proton hopping. The observed experimental and simulated acid concentration dependence then allow us to extrapolate the measured single step proton hopping time to the dilute limit, which, within error, gives the same value as inferred from measurements of the proton mobility and NMR line width analysis. In addition to obtaining the proton hopping time in the dilute limit from direct measurements and AIMD simulations, the results indicate that proton hopping in dilute acid solutions is induced by the concerted multi-water molecule hydrogen bond rearrangement that occurs in pure water. This proposition on the dynamics that drive proton hopping is confirmed by a combination of experimental results from the literature.

Mechanistic Insights into Enzymatic Nitric Oxide Reduction Revealed By Surface-Enhanced Infrared Absorption Spectroscopy

Biological nitric oxide reduction occurs in certain bacteria containing nitric oxide reductases (NORs). The NOR is a transmembrane metalloenzyme and selectively catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N2O) in the microbial denitrification process. The enzymatic NO reduction is catalyzed at a binuclear active center of the non-heme FeB and the heme b 3.[1] The molecular mechanism of the enzymatic NO reduction has been extensively studied using physicochemical techniques including protein film electrochemistry.[2] However, the molecular mechanism still remains under debate. Herein, we report electrochemical and spectroscopic studies on protein film electrochemistry and surface-enhanced infrared absorption (SEIRA) spectroscopy of NOR-modified gold electrodes. The NOR was isolated from P. aeruginosa and then immobilized on SEIRA-active gold electrodes via a self-assembled monolayer of alkyl thiols. The NOR-modified gold electrode showed electrocatalytic NO reduction currents with an onset potential of ca. –0.4 V vs. SHE. Potential-dependent SEIRA spectra of the NOR-modified electrode under CO, which was used as a vibrational probe to identify the electrochemical reduction of iron cofactors, showed a characteristic band at 1972 cm–2 at –0.4 V vs. SHE. This band was associated with the CO binding to heme b 3. Thus, these results suggest that the heme b 3 should be involved in the enzymatic NO reduction.[3] SEIRA spectra of the NOR-modified electrode in the presence of NO are underway to understand the binding mode of NO to iron cofactors in the electrocatalytic condition.

Origin of the piezochromism in Cs2CuCl4 : Electron-phonon and crystal-structure correlations

By using optical absorption and Raman spectroscopy, we have investigated the effects of pressure in the structural, electronic, and vibrational properties of ${\mathrm{Cs}}_{2}{\mathrm{CuCl}}_{4}$ in the 0--20 GPa range. Our study has provided a complete characterization of the electronic and vibrational structures of ${\mathrm{Cs}}_{2}{\mathrm{CuCl}}_{4}$ in the low-pressure Pnma phase as a function of cell volume and the Cu-Cl bond length, ${R}_{\text{Cu-Cl}}$. We have focused on the electronic structure in the Pnma phase, which is mainly related to the tetrahedral ${\mathrm{CuCl}}_{4}^{2\ensuremath{-}}$ units distorted by the Jahn-Teller effect. The strong piezochromism of ${\mathrm{Cs}}_{2}{\mathrm{CuCl}}_{4}$ is caused by the opposite shift of the charge-transfer band gap and the ${\mathrm{Cu}}^{2+}$ d-d crystal-field transitions with pressure. The high-pressure structure of ${\mathrm{Cs}}_{2}{\mathrm{CuCl}}_{4}$ above 4.9 GPa yields structural transformations that we ascribe to a change of coordination around ${\mathrm{Cu}}^{2+}$. The high-pressure phase appears largely amorphized, therefore any structural information from x-ray diffraction is ruled out. Here we use electronic and vibrational probes to get inside the structural information.

Selective excitation of vibrational modes and probe for asymmetric intramolecular energy redistribution

Femtosecond time- and frequency-resolved coherent anti-Stokes Raman scattering spectroscopy was performed on liquid nitrobenzene (NB) to explore intramolecular vibrational energy redistribution in the electronic ground state. While high-frequency C–H stretching vibrational modes (vCH ∼ 3080 cm−1) were selectively excited, vibrational energy redistribution to the nitro group and phenyl ring breathing modes ( ∼ 1335 cm−1, ∼ 1514 cm−1 and ∼1584 cm−1) was described. While lower frequency modes of the nitro group and phenyl ring breathing were selectively excited, vibrational energy redistribution to C–H stretching modes with higher efficiency was detected. There is counter-intuitive vibrational energy flow in NB. Vibrational energy redistribution was discussed in the view of coherent population transfer between parent modes. Coherent coupling of these relevant modes and the selectivity of vibrational energy redistribution were further analyzed from the viewpoint of vibrational symmetry.

Fast dynamics of a hydrogen-bonding glass forming liquid: Chemical exchange-induced spectral diffusion in 2D IR spectroscopy.

Polarization-selective Two Dimensional Infrared (2D IR) and IR pump-probe spectroscopies have been performed on the hydrogen bonding glass forming liquid 2-biphenylmethanol doped with the long-lived vibrational probe phenylselenocyanate over a wide range of temperatures. The spectral diffusion seen in the 2D spectra was found to have a large polarization dependence, in large excess of what is predicted by standard theory. This anomaly was explained by decomposing the 2D spectra into hydrogen-bonding and non-bonding components, which exchange through large-angle orientational motion. By adapting chemical exchange theories, parameters for the component peaks were then calculated by fitting the polarization-dependent spectral diffusion and the pump-probe anisotropy. A model of highly heterogeneous exchange and orientational dynamics was used to explain the observed time dependences as a function of temperature on fast time scales. The experimental observations, the kinetic modeling, and physical arguments lead to the determination of the times for interconversion of slow dynamics structural domains to fast dynamics structural domains in the supercooled liquid as a function of temperature. The slow to fast domain interconversion times range from 40 ps at 355 K to 5000 ps at 270 K.

Imidazole and 1-Methylimidazole Hydrogen Bonding and Nonhydrogen Bonding Liquid Dynamics: Ultrafast IR Experiments.

The dynamics of imidazole (IM) and 1-methylimidazole (1-MeIM) in the liquid phase at 95 °C were studied by IR polarization selective pump-probe and two-dimensional IR (2D IR) spectroscopies. The two molecules are very similar structurally except that IM can be simultaneously a hydrogen bond donor and acceptor and therefore forms extended hydrogen-bonded networks. The broader IR absorption spectrum and a shorter vibrational lifetime of the vibrational probe, selenocyanate anion (SeCN-), in IM vs 1-MeIM indicate that stronger hydrogen bonding exists between SeCN- and IM. Molecular dynamics (MD) simulations support the strong hydrogen bond formation between SeCN- and IM via the HN moiety. SeCN- makes two H-bonds with IM; it is inserted in the IM H-bonded chains rather than being a chain terminator. The strong hydrogen bonding influenced the reorientation dynamics of SeCN- in IM, leading to a more restricted short time angular sampling (wobbling-in-a-cone). The complete orientational diffusion time in IM is 1.7 times slower than in 1-MeIM, but the slow down is less than expected, considering the 3-fold larger viscosity of IM. The jump reorientation mechanism accounts for the anomalously fast orientational relaxation in IM, and the MD simulations determined the average jump angle of the probe between hydrogen bonding sites. Spectral diffusion time constants obtained from the 2D IR experiments are only modestly slower in IM than in 1-MeIM in spite of the significant increase in viscosity. The results indicate that the spectral diffusion sensed by the SeCN- has IM hydrogen bond dynamics contributions not present in 1-MeIM.

The Placement of Vibrational Probe Labeled Substrates to the Phosphopantetheine Arm of the E.Coli Acyl Carrier Protein for Site Specific Vibrational Spectroscopy

The study of acyl carrier proteins (ACP) can provide unique insights into bacterial bio-synthetic pathways due to their integral role of ferrying intermediate substrates to desired enzymes’ active sites. Understanding bacterial biosynthesis can broaden our abilities to synthesis unnatural bioproducts using the cell's machinery. e.Coli's fatty acid ACP is a model for understand the broad family of carrier proteins. One centrally important but poorly understood feature of ACPs is how they protect and present their growing substrates via “chain flipping”. Substrates are placed at the end of a phosphopantetheine (Ppant) arm of holo ACPs and depending on the carbon chain length, the arm can sequester itself in a hydrophobic cavity preventing degradation. Vibrational (infrared and Raman) spectroscopy is an extremely attractive ways to study this process due to the short time scale on which they operate. Vibrational probes of particular interest include isonitriles (which have a very strong, unique IR peak at 2150 cm-1), azides, and alkynes. Recent work points to a modified SFP ligation reaction as a highly effective and versatile method of attaching probe-labeled CoA derivatives to apo ACPs: the challenge is to synthesize the probe-labeled CoA's, which we have attempted using transamidation chemistry. Another, convergent strategy that we have adopted is to incorporate probes within the hydrophobic cavity of the ACP itself via the use of unnatural amino acids. Results from both strategies will be presented. Coupled with the placement of vibrational probes at the end of the Ppant arm, vibrational spectroscopic experiments can offer a well rounded image of ACP's dynamic nature as it interacts with itself and its catalytic partners.

P-Nitrophenylalanine as a Novel Probe of Hydrogen Bonding in Peptide Hydrogels and Proteins

Using vibrational probes to report on the local environment of specific protein residues has became very popular in recent years. Aromatic nitro groups are easily incorporated into peptides and proteins, and the vibrational modes of the nitro groups have been proposed as IR (or Raman) probes. Currently, quantitative data on how the nitro group's frequencies respond to the local environment is unclear. Previous IR work led to focus on a mode near 1600 cm-1. Now, we see a very strong Raman scattering peak at about 1350 cm-1: calculations indicate that this comes from a normal mode that contains symmetric nitro and ring motions. We used the compound 4-nitro-benzyl alcohol and a home built Raman spectrometer to determine the solvatochromism of this band in a wide range of solvents. Our results showed that strong hydrogen bond donor solvents led to a blue shift in the frequency of this Raman band, and a linear regression analysis using empirical solvent scales indicated that H-bond donors are the main factor that determines the nitro stretching frequency. Using this new information, we placed p-nitrophenylalanine as a Raman probe in a hydrogel-forming peptide derived from semenogelin I. Our results showed that the peptides stayed intact and the probe was clearly visible across different concentrations in both H2O and D2O, with different frequencies in three different positions where p-nitrophenylalanine replaced either phenylalanine or tyrosine residues along what appears to be a hydrophobic cluster that holds together the hydrogel's beta sheet superstructure. P-nitrophenylalanine was also incorporated into the E. Coli acyl carrier protein at key sites using the stop codon suppression technique. In all peptide and protein samples, this band is visible despite its spectral overlap with other vibrations.

Using Site-Specific Vibrational Spectroscopy to Determine Structure of Membrane-Bound N-Terminally Acetylated Alpha-Synuclein

Despite a demonstrated connection to the etiology of Parkinson's Disease (PD), the structure and function of the neuronal brain protein alpha-synuclein (aS) remain elusive and confuse attempts to elucidate the link between the protein and PD. Structure determination is difficult because aS is an intrinsically-disordered protein in solution and because the recently ubiquity of N-terminal acetylation in vivo questions the relevance of many previous experiments that used unacetylated aS. Most previous research on aS membrane-bound structure, primarily using NMR, also lacks the ability to directly assess conformational dynamics on very short timescales. Site-specific vibrational spectroscopy presents a new technique that could reveal new details of the membrane-bound conformational distribution of aS. Here, we first attempted to obtain N-terminally acetylated aS (Nt-Ac aS) via the coexpression of human aS and yeast N-acetyltransferase B (NatB) in E. coli cells but failed to complete the acetylation. An adapted expression protocol using a sequence of increasing-volume cultures (according to the lab of David Eliezer) and E. coli cells pretransformed to express NatB before transformation of aS (according to the lab of Elizabeth Rhoades) successfully yielded several single-cysteine mutants of N-terminally acetylated aS, and we converted the thiol to the SCN vibrational probe group. We used infrared spectroscopy to measure solvent exposure of the SCN group at several sites along membrane-bound unacetylated and acetylated aS and compared the results. SCN-labeled variants of aS should be useful to explore aS's membrane-bound conformations in contact with a wide variety of lipid systems.

Research progress of molecular structure and dynamics of biological water

The specific water molecules that are confined within the solvation shell adjacent to the surface of biological macromolecules (including protein, enzyme, DNA, RNA, cell membrane, etc.) are called biological water molecules. Such water around the biomolecule surface plays a very important role in the structure, stability, dynamics, and function of biological macromolecules. A molecular-level understanding of the structure and dynamics of biological water, as well as the nature of its influence on biological structure and function is the key to revealing the mechanism of the biological functions. However, the researches in this field are still in the initial stage. Here in this paper, we review the relevant researches and recent progress of hydration water from three aspects. The first aspect is about the influence of hydration water on biological structure and function. It is evident that water actively participates in many biological processes such as protein folding, proton donation and migration, ligand binding and drug design, and allosteric effects. For example, water mediates the collapse of the chain and the search for the native topology through a funneled energy landscape. The second aspect is about the structure of water molecules around the biomolecules investigated by nuclear magnetic resonance (NMR), dielectric relaxation, neutron scattering, X-ray diffraction and ultrafast optical spectroscopy. The third aspect is about the dynamic behaviors of biological water, including the relaxation time scale, dynamic property, dynamic coupling between biomolecules and water molecules, and sub-diffusive motion of the water molecules along the protein surfaces. Different techniques measure different timescales for the motion of proteins and their hydration environment. While NMR and dielectric relaxation methods reveal the motion of biological water on a time scale from several tens of picoseconds to nanoseconds, ultrafast optical spectroscopy such as fluorescence and vibrational spectroscopy probes the hydrogen-bonding fluctuations of water on a time scale from the femtosecond to picosecond. It is therefore highly necessary to acquire a real and complete picture of the structure and dynamics of biological water by combining several different techniques. Finally, some unsolved scientific problems are also summarized in this review.

Counterion Effect on Vibrational Relaxation and the Rotational Dynamics of Interfacial Water and an Anionic Vibrational Probe in the Confined Reverse Micelles Environment

Vibrational relaxation and the rotational dynamics of water molecules encapsulated in reverse micelles (RMs) have been investigated by ultrafast infrared (IR) spectroscopy and two-dimensional IR (2D IR) spectroscopy. By changing the counterion of the hydrophilic headgroup in the RMs formed by Aerosol-OT (AOT) from Na+ to K+, Cs+ and Ca2+, we could determine the specific counterion effects on the rotational dynamics of water molecules. The orientational relaxation time constant of water decreases in the order Ca2+ > Na+ > K+ > Cs+. The SCN- anionic probe and counterion can form ion pairs at the interfacial region of the RMs. The rotational dynamics of SCN- anion significantly decreases because of the synergistic effects of confinement and the surface interactions in the interfacial region of the RMs. The results can provide a new understanding of the cationic Hofmeister effect at the molecular level observed in biological studies.

Structural Dynamics of Dimethyl Sulfoxide Aqueous Solutions Investigated by Ultrafast Infrared Spectroscopy: Using Thiocyanate Anion as a Local Vibrational Probe.

The microscopic structure of dimethyl sulfoxide (DMSO) aqueous solutions was investigated by Fourier transform infrared (FTIR) spectroscopy and ultrafast IR spectroscopy. The structural dynamics of the binary mixtures were reflected by using thiocyanate anion (SCN-) as a local vibrational probe. FTIR spectra of SCN- anion showed that the hydrogen bond networks of water are affected by the presence of DMSO molecules, and the peak position and bandwidth of SCN- anions are red shifted and narrowed accordingly because of the weak hydration in the binary mixture. The vibrational lifetime of the SCN- anion showed almost linear enhancement with the increase of DMSO, which can be explained by the weak interaction between SCN- and the hydrophobic groups in the DMSO molecule. However, the rotational dynamics of SCN- are slowing down significantly and showed a maximum response at XDMSO (mole fraction) of 0.35, which is mainly caused by the confinement of SCN- anions positioned in the vicinity of the complex structure formed between DMSO and water molecules. The concentration-dependent rotational dynamics of water molecules and SCN- anions are having similar behavior, indicating that the complex structure can be formed between water and DMSO molecules because of the strong interaction. The result also demonstrates that the structural inhomogeneity in aqueous solution can be unraveled by monitoring the vibrational relaxation dynamics of SCN- anion serving as the local vibrational probe.

Quantifying conformations of ester vibrational probes with hydrogen-bond-induced Fermi resonances.

Solvatochromic shifts of local vibrational probes report on the strength of the surrounding electric fields and the probe's hydrogen bonding status. Stretching vibrational mode of the ester carbonyl group is a popular solvatochromic reporter used in the studies of peptides and proteins. Small molecules, used to calibrate the response of the vibrational probes, sometimes involve Fermi resonances (FRs) induced by inter-molecular interactions. In the present work, we focus on the scenario where FR does not appear in the infrared spectrum of the ester carbonyl stretching mode in aprotic solvents; however, it is intensified when a hydrogen bond with the reporter is established. When two molecules form hydrogen bonds to the same carbonyl oxygen atom, FR leads to strong hybridization of the involved modes and splitting of the absorption peak. Spectral overlap between the Fermi doublets associated with singly and doubly hydrogen-bonded carbonyl groups significantly complicates quantifying different hydrogen-bonded conformations. We employed a combination of linear and third-order (2DIR) infrared spectroscopy with chemometrics analysis to reveal the individual line shapes and to estimate the occupations of the hydrogen-bonded conformations in methyl acetate, a model small molecule. We identified a hydrogen-bond-induced FR in complexes of methyl acetate with alcohols and water and found that FR is lifted in larger molecules used for control experiments-cholesteryl stearate and methyl cyanoacetate. Applying this methodology to analyze acetonitrile-water solutions revealed that when dissolved in neat water, methyl acetate occupies a single hydrogen-bonding conformation, which is in contrast to the conclusions of previous studies. Our approach can be generally used when FRs prevent direct quantification of the hydrogen bonding status of the vibrational probe.

Conformation-specific detection of calmodulin binding using the unnatural amino acid p-azido-phenylalanine (AzF) as an IR-sensor.

Calmodulin (CaM) is a very conserved, ubiquitous, eukaryotic protein that binds four Ca2+ ions with high affinity. It acts as a calcium sensor by translating Ca2+ signals into cellular processes such as metabolism, inflammation, immune response, memory, and muscle contraction. Calcium binding to CaM leads to conformational changes that enable Ca2+/CaM to recognize and bind various target proteins with high affinity. The binding mode and binding partners of CaM are very diverse, and a consensus binding sequence is lacking. Here, we describe an elegant system that allows conformation-specific detection of CaM-binding to its binding partners. We incorporate the unnatural amino acid p-azido-phenylalanine (AzF) in different positions of CaM and follow its unique spectral signature by infrared (IR)-spectroscopy of the azido stretching vibration. Our results suggest that the AzF vibrational probe is sensitive to the chemical environment in different CaM/CaM-binding domain (CaMBD) complexes, which allows differentiating between different binding motifs according to the spectral characteristics of the azido stretching mode. We corroborate our results with a crystal structure of AzF-labelled CaM (CaM108AzF) in complex with a binding peptide from calmodulin-dependent protein kinase IIα identifying the structural basis for the observed IR frequency shifts.

How Molecular Crowding Differs from Macromolecular Crowding: A Femtosecond Mid-Infrared Pump-Probe Study.

Crowding is an inherent property of living systems in which biochemical processes occur in highly concentrated solutions of various finite-sized species of both low (molecular crowding) and high (macromolecular crowding) molecular weights. Is molecular crowding fundamentally different from macromolecular crowding? To answer this question, we use a femtosecond mid-infrared pump-probe technique with three vibrational probes in molecular (diethylene glycol) and macromolecular (polyethylene glycol) solutions. In less crowded media, both molecular and macromolecular crowders fail to affect the dynamics of interstitial bulk-like water molecules and those at the crowder/water interface. In highly crowded media, interstitial water dynamics strongly depends on molecular crowding, but macromolecular crowding does not alter the bulk-like hydration dynamics and has a modest crowding effect on water at the crowder/water interface. The results of this study provide a molecular level understanding of the structural and dynamic changes to water and the water-mediated cross-linking of crowders.