IR Probes Research Articles

In situ X-ray and IR probes relevant to Earth science at the Advanced Light Source at Lawrence Berkeley Laboratory

Access to synchrotron X-ray facilities has become an important aspect for many disciplines in experimental Earth science. This is especially important for studies that rely on probing samples in situ under natural conditions different from the ones found at the surface of the Earth. The non-ambient condition Earth science program at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory, offers a variety of tools utilizing the infra-red and hard X-ray spectrum that allow Earth scientists to probe Earth and environmental materials at variable conditions of pressure, stress, temperature, atmospheric composition, and humidity. These facilities are important tools for the user community in that they offer not only considerable capacity (non-ambient condition diffraction) but also complementary (IR spectroscopy, microtomography), and in some cases unique (Laue microdiffraction) instruments. The availability of the ALS’ in situ probes to the Earth science community grows especially critical during the ongoing dark time of the Advanced Photon Source in Chicago, which massively reduces available in situ synchrotron user time in North America.

Infrared Spectroscopy of Liquid Solutions as a Benchmarking Tool of Semiempirical QM Methods: The Case of GFN2-xTB.

The accurate description of large molecular systems has triggered the development of new computational methods. Due to the computational cost of modeling large systems, the methods usually require a trade-off between accuracy and speed. Therefore, benchmarking to test the accuracy and precision of the method is an important step in their development. The typical gold standard for evaluating these methods is isolated molecules, because of the low computational cost. However, the advent of high-performance computing has made it possible to benchmark computational methods using observables from more complex systems such as liquid solutions. To this end, infrared spectroscopy provides a suitable set of observables (i.e., vibrational transitions) for liquid systems. Here, IR spectroscopy observables are used to benchmark the predictions of the newly developed GFN2-xTB semiempirical method. Three different IR probes (i.e., N-methylacetamide, benzonitrile, and semiheavy water) in solution are selected for this purpose. The work presented here shows that GFN2-xTB predicts central frequencies with errors of less than 10% in all probes. In addition, the method captures detailed properties of the molecular environment such as weak interactions. Finally, the GFN2-xTB correctly assesses the vibrational solvatochromism for N-methylacetamide and semiheavy water but does not have the accuracy needed to properly describe benzonitrile. Overall, the results indicate not only that GFN2-xTB can be used to predict the central frequencies and their dependence on the molecular environment with reasonable accuracy but also that IR spectroscopy data of liquid solutions provide a suitable set of observables for the benchmarking of computational methods.

A disparity in solvatochromism of C[dbnd]O and S[dbnd]O vibrational probe: A study of structurally similar acetone and dimethyl sulfoxide

Solvatochromic studies of small IR probes are beneficialto study the structural dynamics of biomolecules as well as measuring the strength of surrounding electric fields in condensed phases.Herein, we have investigated the CO and SO vibrational probes of structurally similar acetone and dimethyl sulfoxide respectively to compare the solvatochromism. Interestingly in water CO formed both one hydrogen bond and two hydrogen bond complexes wherein SO formed only two hydrogen bond complexes.Not only this, but we have also investigated how different solvents alter the IR spectra of the CO and SO vibrating modes during the interaction with α CH of acetone and DMSO respectively.The IR absorption spectra of CO of acetone altered after αdeuteration(acetoned6) in aprotic organic solvents as well as aqueous solvents. But, surprisingly for DMSO and DMSOd6, the IR spectra of SO mode remain intact during α isotopic exchange (CH3to CD3) in organic aprotic solvents but in strong coordinating polar protic solvents (D2O) it is altered remarkably. The equivalent frequency gap of CO of acetone and acetoned6 in organic solvents and water represent the no interaction of α CH/D with solvent molecules. Parallelly unaltered IR spectral signature of SO of both DMSO and DMSOd6 in aprotic organic solvent represents the stationary state of the ‘S’ atom and altered SO IR absorption in water signifies the different α CH and α C-D interaction with the surrounding water molecules. These results are the cautionary of perturbation on IR probe during α CH interaction with solvents as well as guest molecules.

Dynamic Ir(III) Photosensors for the Major Human Drug-Metabolizing Enzyme Cytochrome P450 3A4.

Probing the activity of cytochrome P450 3A4 (CYP3A4) is critical for monitoring the metabolism of pharmaceuticals and identifying drug-drug interactions. A library of Ir(III) probes that detect occupancy of the CYP3A4 active site were synthesized and characterized. These probes show selectivity for CYP3A4 inhibition, low cellular toxicity, Kd values as low as 9 nM, and are highly emissive with lifetimes up to 3.8 μs in cell growth media under aerobic conditions. These long emission lifetimes allow for time-resolved gating to distinguish probe from background autofluorescence from growth media and live cells. X-ray crystallographic analysis revealed structure-activity relationships and the preference or indifference of CYP3A4 toward resolved stereoisomers. Ir(III)-based probes show emission quenching upon CYP3A4 binding, then emission increases following displacement with CYP3A4 inhibitors or substrates. Importantly, the lead probes inhibit the activity of CYP3A4 at concentrations as low as 300 nM in CYP3A4-overexpressing HepG2 cells that accurately mimic human hepatic drug metabolism. Thus, the Ir(III)-based agents show promise as novel chemical tools for monitoring CYP3A4 active site occupancy in a high-throughput manner to gain insight into drug metabolism and drug-drug interactions.

Development of temperature-responsive suspension stabilizer and its application in cementing slurry system

To solve the problem of cement slurry suspension instability caused by a geothermal gradient in low-pressure and leakage formation, a hydrophobically associating long-chain polymer (PSAN) was designed as a suspension stabilizer by introducing hydrophilic monomer acrylamide (AM), temperature-resistant monomer 2-acrylamide-2-methyl propane sulfonic acid (AMPS), hydrophobic monomer styrene (St) and temperature-sensitive monomer N-vinyl caprolactam (NVCL). The synthesis conditions of PSAN were optimized by orthogonal response surface method. The structure and performance of PSAN were characterized and tested by IR, 1H NMR, GPC, SEM, TG, high-temperature rheology, and fluorescent probes. The research and analysis showed that PSAN had a good temperature response. At 30–65 ℃, the apparent viscosity of PSAN aqueous solution increased with the increase of temperature. At 65 ℃, the hydrophobically associating ability of PSAN was enhanced, and the retention rate of the apparent viscosity of its aqueous solution at 65–110 ℃ was still more than 83%. The good temperature response of PSAN was conducive to the suspension stability of cement slurry under the condition of large temperature differences. When 1% bwoc (percentage of cement mass) was added to the cement slurry, the free liquid of the cement slurry was 0, the water loss was less than 90 mL, and the density difference between the upper and lower sections of the cement stone column was less than 0.01 g/cm3. SEM analysis showed that there was a network structure formed by hydrophobic association between PSAN molecules in cement. In a word, under the condition of 30–110 ℃ (80 ℃ large temperature difference), the suspension stabilizer PSAN can effectively alleviate the floating of light materials and the sinking of heavy materials in cementing slurry through the hydrophobic association between molecules, and ensure the suspension stability of cementing slurry, thus contributing to the safety of cementing construction and improving the quality of cementing.

Direct Probing of Fe3O4 Nanoparticle Surface Temperatures during Magnetic Heating: Implications for Induction Catalysis

The presence of a temperature gradient between the magnetic nanoparticle (NP) surface and the bulk medium during induction heating has been observed across several fields. While no noticeable increase in bulk temperature is observed, biological (DNA denaturation, tumor apoptosis) and chemical (bond cleavage) evidence indicates high temperatures near/at the NP surface. Unfortunately, current methods for temperature probing rely on bulk temperature measurements (fiber-optic IR probes) or are limited by thermal stability and spatial resolution (organic molecules). To further the understanding of magnetic heating as a driving force in catalysis, as well as drug delivery/hyperthermia treatments, a more accurate description of the nanoparticle surface temperature is needed. This work uses inorganic luminescent probes in direct contact with the particle surface, entailing the deposition of YVO4:Eu3+ around a Fe3O4|SiO2 structure, to measure the local temperature. The luminescent response is calibrated in situ via a controlled temperature stage to extract the field-dependent heating. The luminescent probe results in a high spatial resolution (<5.5 nm) with temperatures up to 64 °C higher than standard fiber-optic probes. The direct contact between the photoluminescence (PL) probe and Fe3O4 allows for ballistic transport and improved temporal resolution, mimicking an adiabatic system (negligible long-range heat dissipation). Other advantages include avoiding measurements in liquid media, where the distance between the heat source and the probe cannot be controlled, adding to the uncertainty of the temperature measurement due to changes in colloidal anisotropy (which changes with the heating profile) of the magnetic cores and surface quenching of the luminescent signal.

Substituent Effects on the Vibrational Properties of the CN Stretch Mode of Aromatic Nitriles: IR Probes Useful for Time-resolved IR Spectroscopy.

Developing ideal IR probes is essential to understand the structure and dynamics of biomolecules with time-resolved IR spectroscopies and imaging techniques. Especially, nitrile (CN) group has recently been proposed to serve as IR probes of the local environment of proteins. Herein, we investigated the effect of a substituent on the vibrational properties of the benzonitrile. The electron-donating and withdrawing character of p-substituent on benzonitrile are expected to modulate the vibrational frequency, molar extinction coefficient, and vibrational lifetime of CN probe. FT-IR revealed the positive correlation between electron-donating character and the molar extinction coefficient of CN stretch mode. Infrared pump-probe (IR-PP) measurements showed that the vibrational lifetime of CN stretch mode exhibits a relatively weak correlation with the electron-donating strength. Among the investigated samples, 4-dimethylamino benzonitrile with the strongest electron-donating strength shows enhanced absorption and extended vibrational lifetime. Utilizing substituent effects will be a practical strategy to improve the performance of the IR probe.

Room-Temperature Synthesis of a Hollow Microporous Organic Polymer Bearing Activated Alkyne IR Probes for Nonradical Thiol-yne Click-Based Post-Functionalization.

This work shows that hollow microporous organic polymer (H-MOP-A) with activated internal alkynes as IR probes can be prepared by template synthesis based on acyl Sonogashira-Hagihara coupling at room temperature. The H-MOP-A is a versatile platform in the main chain PSM based on nonradical thiol-yne click reaction. Moreover, an IR peak of internal alkynes in the H-MOP-A is very intense and could be utilized in the monitoring of thiol-yne click-based main chain PSM. The functionalized H-MOP-A with carboxylic acids (H-MOP-CA) showed efficient adsorption toward Ag+ ions. The resultant H-MOP-CA-Ag showed excellent performance in the CO2 fixation to α-alkylidene cyclic compounds.

Development of iridium(III) phosphorescent probe for hypochlorous acid detection in macrophages cells and cancer cells co-culture system and application in inflamed mouse model

Macrophages infiltrated in tumor microenvironment (TME) were found to play vital roles in immune responses and tumor development. In the process of immune, macrophages could produce pro-inflammatory mediators including ROS. Thus, targeting macrophages related ROS and distinguishing macrophages and cancer cells in one or more types of tumor and tumor microenvironments is extremely important and necessary to better understand tumor-associated macrophages (TAMs) functions and provide new insights into macrophage-centred therapeutic. Herein, we developed two unique Ir(III) probes, Ir1 and Ir2, for HOCl detection. Because of the dual protection of C^N ligand, Ir2 exhibits high sensitivity and selectivity in the present of HOCl. Therefore, Ir2 can be employed to detect HOCl in flow cytometry and image exogenous and endogenous HOCl in mitochondria. Importantly, Ir2 was applied to distinguish macrophages cells and cancer cells in TAMs by flow cytometry. Moreover, Ir2 is applicable in staining five kinds of organs of mouse tissues and monitoring HOCl generation in the LPS-stimulated inflammation of mouse model with two-photon microscopy (TPM).

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.

Simultaneous enhancement of transition dipole strength and vibrational lifetime of an alkyne IR probe viaπ-d backbonding and vibrational decoupling.

Alkyne infrared (IR) probes 1-6 with Si and S (or Se) atoms incorporated into the C[triple bond, length as m-dash]C bond were synthesized, and the vibrational properties of their C[triple bond, length as m-dash]C stretch mode were studied using Fourier transform infrared (FTIR) and femtosecond IR pump-probe (IR PP) spectroscopies in combination with quantum chemical calculations. From FTIR studies, the transition dipole strengths (in units of 10-2 D2) of 1-3 having the Si atom were measured to be 1.85, 3.32, and 2.52, whereas those of 4-6 having no Si atom were measured to be 0.13, 0.20, and 0.17, respectively, in CHCl3. Thus, the increase in the transition dipole strength of the C[triple bond, length as m-dash]C stretch mode upon incorporation of the Si atom into the C[triple bond, length as m-dash]C bond is by a factor of about 14 or higher. The large increase in the transition dipole strength of the C[triple bond, length as m-dash]C stretch mode upon such Si incorporation is attributed to π-d backbonding between the C[triple bond, length as m-dash]C group's π and Si atom's d orbitals. From IR PP experiments, the vibrational lifetimes of the C[triple bond, length as m-dash]C stretch mode in 1-3 having none, S, and Se atoms were determined to be 5.7 ± 0.7, 13.0 ± 1.1, and 94.2 ± 5.8 ps, respectively, in CHCl3. Thus, the increase in the vibrational lifetime of the C[triple bond, length as m-dash]C stretch mode upon incorporation of the S (or Se) atom between the phenyl ring and the C[triple bond, length as m-dash]C bond is by a factor of about 2 (or 16) or higher. The large increase in the vibrational lifetime of the C[triple bond, length as m-dash]C stretch mode upon such S (or Se) incorporation is attributed to its heavy atom effect impeding vibrational couplings between the C[triple bond, length as m-dash]C stretch and phenyl ring vibrations. From two-dimensional infrared (2DIR) experiments, the large transition dipole strength and long vibrational lifetime of 3 containing the Si and S (or Se) atoms were shown to enable the measurement of its 2DIR spectra up to 500 ps. The strongly absorbing alkynes with long vibrational lifetimes will be a promising probe of molecular dynamics in nonlinear vibrational spectroscopy and imaging on an extended time scale.

High‐Performance Red/Near‐IR Carbon Dots as Fluorescence Probes for Tumor Imaging In Vivo

AbstractCarbon dots (CDots) are brightly fluorescent, especially in the same green spectral region overlapping that of the green fluorescent protein, and also benign and nontoxic. For imaging in vivo, however, red/near‐IR probes are required for the high optical transmittance through tissues and the contrast against interference from autofluorescence background. Herein, high‐performance red/near‐IR CDots are designed and prepared by modifying the core carbon nanoparticles in CDots with the incorporation of selected dye species into the nanoparticle structure. The representative modified CDots thus obtained are used as fluorescence probes in the in vivo imaging of normal and tumor‐bearing nude mice via various exposure pathways. The results suggest that these red/near‐IR CDots are indeed excellent probes for fluorescence imaging in vivo, especially with their enhanced tumor uptake. The potentially far‐reaching implications of being able to design and synthesize the modified CDots of strong optical absorptions and bright fluorescence emissions in the red/near‐IR are discussed in terms of their broad applicability to in vivo imaging and theranostics.

Multipurpose isothiocyanyl alanine/lysine: Use as solvatochromic IR probes and in site specific labeling/ligation of short peptides

The solvatochromic IR responsivity of small side chain –NCS in two unexplored unnatural amino acids, isothiocyanyl alanine (NCSAla = Ita) and lysine (NCSLys = Itl), without perturbing the conformation is demonstrated in two designed short tripeptide (BocAla-NCSAla-Ala-OMe) and hexapeptide (BocLeu-Val-Phe-Phe-NCSLys-Gly-OMe). Demonstration of site specific fluorescent labeling in both the peptides and ligation type reaction in NCSLys indicates the novelty of these two amino acids as alternative to the available canonical amino acids.

Effect of Osmolytes on the Conformational Behavior of a Macromolecule in a Cytoplasm-like Crowded Environment: A Femtosecond Mid-IR Pump-Probe Spectroscopy Study.

Osmolytes found endogenously in almost all living beings play an important role in regulating cell volume under harsh environment. Here, to address the longstanding questions about the underlying mechanism of osmolyte effects, we use femtosecond mid-IR pump-probe spectroscopy with two different IR probes that are the OD stretching mode of HDO and the azido stretching mode of azido-derivatized poly(ethylene glycol) dimethyl ether (PEGDME). Our experimental results show that protecting osmolytes bind strongly with water molecules and dehydrate polymer surface, which results in promoting intramolecular interactions of the polymer. By contrast, urea behaves like water molecules without significantly disrupting water H-bonding network and favors extended and random-coil segments of the polymer chain by directly participating in solvation of the polymer. Our findings highlight the importance of direct interaction between urea and macromolecule, while protecting osmolytes indirectly affect the macromolecule through enhancing the water-osmolyte interaction in a crowded environment, which is the case that is often encountered in real biological systems.

Evaluation of p-(13C,15N-Cyano)phenylalanine as an Extended Time Scale 2D IR Probe of Proteins.

Two-dimensional infrared (2D IR) spectroscopy provides a powerful approach for the direct study of molecular dynamics with high spatial and temporal resolution. Its application for investigating specific locations in proteins requires the incorporation of IR probe groups with spectrally isolated absorptions to avoid the congestion inherent to protein spectra. This has motivated extensive efforts toward the development of new IR probes, but there remains a need for those that can extend the experimental time range, which is limited by their vibrational lifetimes. Toward this goal, isotopically labeled p-(13C15N-cyano)phenylalanine was synthesized, site-selectively incorporated into the protein plastocyanin, and evaluated for its potential as a 2D IR probe. The isotopic labeling increases the vibrational lifetime about 2-fold, which results in larger signals at longer time scales. However, isotopic labeling simultaneously shifts the absorption to a spectral region with greater water absorbance, which results in greater heating-induced signals in the background that overlap those of the nitrile probe. The study demonstrates the use of a new 2D IR probe to measure the side chain dynamics in a protein and also illustrates the multiple factors to consider in development of 2D IR probes for studying proteins.

Vibrational Probes: From Small Molecule Solvatochromism Theory and Experiments to Applications in Complex Systems.

The vibrational frequency of a chosen normal mode is one of the most accurately measurable spectroscopic properties of molecules in condensed phases. Accordingly, infrared absorption and Raman scattering spectroscopy have provided valuable information on both distributions and ensemble-average values of molecular vibrational frequencies, and these frequencies are now routinely used to investigate structure, conformation, and even absolute configuration of chemical and biological molecules of interest. Recent advancements in coherent time-domain nonlinear vibrational spectroscopy have allowed the study of heterogeneous distributions of local structures and thermally driven ultrafast fluctuations of vibrational frequencies. To fully utilize IR probe functional groups for quantitative bioassays, a variety of biological and chemical techniques have been developed to site-specifically introduce vibrational probe groups into proteins and nucleic acids. These IR-probe-labeled biomolecules and chemically reactive systems are subject to linear and nonlinear vibrational spectroscopic investigations and provide information on the local electric field, conformational changes, site-site protein contacts, and/or function-defining features of biomolecules. A rapidly expanding library of data from such experiments requires an interpretive method with atom-level chemical accuracy. However, despite prolonged efforts to develop an all-encompassing theory for describing vibrational solvatochromism and electrochromism as well as dynamic fluctuations of instantaneous vibrational frequencies, purely empirical and highly approximate theoretical models have often been used to interpret experimental results. They are, in many cases, based on the simple assumption that the vibrational frequency of an IR reporter is solely dictated by electric potential or field distribution around the vibrational chromophore. Such simplified description of vibrational solvatochromism generally referred to as vibrational Stark effect theory has been considered to be quite appealing and, even in some cases, e.g., carbonyl stretch modes in amide, ester, ketone, and carbonate compounds or proteins, it works quantitatively well, which makes it highly useful in determining the strength of local electric field around the IR chromophore. However, noting that the vibrational frequency shift results from changes of solute-solvent intermolecular interaction potential along its normal coordinate, Pauli exclusion repulsion, polarization, charge transfer, and dispersion interactions, in addition to the electrostatic interaction between distributed charges of both vibrational chromophore and solvent molecules, are to be properly included in the theoretical description of vibrational solvatochromism. Since the electrostatic and nonelectrostatic intermolecular interaction components have distinctively different distance and orientation dependences, they affect the solvatochromic vibrational properties in a completely different manner. Over the past few years, we have developed a systematic approach to simulating vibrational solvatochromic data based on the effective fragment potential approach, one of the most accurate and rigorous theories on intermolecular interactions. We have further elucidated the interplay of local electric field with the general vibrational solvatochromism of small IR probes in either solvents or complicated biological systems, with emphasis on contributions from non-Coulombic intermolecular interactions to vibrational frequency shifts and fluctuations. With its rigorous foundation and close relation to quantitative interpretation of experimental data, this and related theoretical approaches and experiments will be of use in studying and quantifying the structure and dynamics of biomolecules with unprecedented time and spatial resolution when combined with time-resolved vibrational spectroscopy and chemically sensitive vibrational imaging techniques.

Studying Water Hydrogen-Bonding Network near the Lipid Multibilayer with Multiple IR Probes.

A critical difference between living and nonliving is the existence of cell membranes, and hydration of membrane surface is a prerequisite for structural stability and various functions such as absorption/desorption of drugs, proteins, and ions. Therefore, a molecular level understanding of water structure and dynamics near the membrane is important to perceive the role of water in such a biologically relevant environment. In our recent paper [ J. Phys. Chem. Lett. 2016 , 7 , 741 ] on the IR pump-probe study of the OD stretch mode of HDO near lipid multibilayers, we have observed two different vibrational lifetime components of OD stretch mode in the phospholipid multibilayer systems. The faster component (0.6 ps) is associated with OD groups interacting with the phosphate moiety of the lipid, while the slower component (1.9 ps) is due to choline-associated water molecules that are close to bulklike water. Here, we additionally use hydrazoic acid (HN3) as another IR probe of which frequency is highly sensitive to its local H-bonding water density. Interestingly, we found that the vibrational lifetime of the asymmetric azido stretch mode of HN3 in the lipid multibilayer system is similar to that in neat water, whereas its orientational relaxation is a bit slower than that in bulk water. This indicates that due to the tight packing of lipid molecules, particularly the head parts, in the gel phase, HN3 molecules mostly stay near the choline group of lipid and interact with water molecules in the vicinity of choline groups. This suggests that membrane surface-adsorbed molecules such as hydrophilic drug molecules may interact with choline-associated water molecules, when the membrane is in the gel phase, instead of phosphate-associated water molecules.

Extended timescale 2D IR probes of proteins: p-cyanoselenophenylalanine.

The importance of dynamics to the function of proteins is well appreciated, but the difficulty in their measurement impedes investigation into their precise role(s). 2D IR spectroscopy is a developing approach for the study of dynamics and has motivated efforts to develop spectrally resolved IR probe groups that enable its application for measuring the dynamics at specific sites in a protein. A challenge with this approach is that the timescales accessible are limited by the vibrational lifetimes of the probes. Toward development of better probes for 2D IR spectroscopy of protein dynamics, we report the characterization of p-cyano-seleno-phenylalanine (CNSePhe), a derivative of the well established IR probe p-cyano-phenylalanine (CNPhe), by FT IR, pump-probe, and 2D IR spectroscopy. The incorporation of the heavy Se atom decouples the CN vibration from the rest in the molecule. Although this leads to a reduction of the transition dipole strength, and thus a reduction in signal intensity, it also dramatically increases the vibrational lifetime, enabling collection of 2D IR spectra for analysis of molecular dynamics on much longer timescales. Interestingly, we also find that the lifetime for CNSePhe shows increased sensitivity to the presence of hydrogen bonding interactions with the CN, suggesting that the probe should be useful for interpretation of CN spectra and possibly for the study of solvation.

Water Dynamics in Cytoplasm-Like Crowded Environment Correlates with the Conformational Transition of the Macromolecular Crowder.

Polyethylene glycol (PEG) is a unique polymer material with enormous applicability in many industrial and scientific fields. Here, its use as macromolecular crowder to mimic the cellular environment in vitro is the focus of the present study. We show that femtosecond mid-IR pump-probe spectroscopy using three different IR probes, HDO, HN3, and azido-derivatized crowder, provides complete and stereoscopic information on water structure and dynamics in the cytoplasm-like macromolecular crowding environment. Our experimental results suggest two distinct subpopulations of water molecules: those that interact with other water molecules and those that are part of a hydration shell of crowder on its surface. Interestingly, water dynamics even in highly crowded environment remains bulk-like in spite of significant perturbation to the tetrahedral H-bonding network of water molecules. That is possible because of the formation of water aggregates (pools) even in water-deficient PEGDME-water solutions. In such a crowded environment, the conformationally accessible phase space of the macromolecular crowder is reduced, similar to biopolymers in highly crowded cytoplasm. Nonetheless, the hydration water on the surface of crowders slows down considerably with increased crowding. Most importantly, we do not observe any coalescing of surface hydration water (of the crowder) with bulk-like water to generate collective hydration dynamics at any crowder concentration, contrary to recent reports. We anticipate that the present triple-IR-probe approach is of exceptional use in studying how conformational states of crowders correlate with structural and dynamical changes of water, which is critical in understanding their key roles in biological and industrial applications.

Temperature Dependence of CN and SCN IR Absorptions Facilitates Their Interpretation and Use as Probes of Proteins

Cyano and thiocyano groups have received attention as IR probes of local protein electrostatics or solvation, due to their strong absorptions and the ability to site specifically incorporate them within proteins. However, interpreting their spectra requires knowing whether they engage in hydrogen bonds (H-bonds). Existing methods for the detection of such H-bonding interactions are based on structural analysis or correlations between IR and NMR signals and are labor intensive and possibly ambiguous. Here, using model systems we show that the absorption frequency of both probes is linearly correlated with temperature and that the slope of the resulting line (frequency-temperature line slope or FTLS) reflects the nature of the probe's microenvironment, including whether or not the probe is engaged in H-bonds. We then show that the same linear dependence is observed with p-cyano phenylalanine, cyanylated cysteine, or cyanylated homocysteine incorporated at different positions within the N-terminal Src homology 3 domain of the murine adapter protein Crk-II. The FTLSs indicate that p-cyano phenylalanine incorporated at two positions is engaged in strong H-bonding, while it is involved in weaker H-bonding at a third position. In contrast, the FTLS of the cyanylated cysteine or cyanylated homocysteine absorptions indicates that they do not engage in H-bonding at either a buried or surface exposed position. While the differences likely reflect side chain flexibility and the probe's ability to avoid solvent, the data suggest that the temperature dependence of the absorption provides a simple method to gauge the probe's environment, including the presence of H-bonding.