Isolobal Cationic Iridium Dihydride and Dizinc Complexes: A Dual Role for the ZnR Ligand Enhances H Activation.

The reaction of [Ir(IPr)H][BAr] (; IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene; BAr = B{CH(3,5-CF)}) with ZnMe proceeds with CH elimination to give [Ir(IPr)(IPr')(ZnMe)H][BAr] (, where (IPr') is a cyclometalated IPr ligand). reacts with H to form tetrahydride [Ir(IPr)(ZnMe)H][BAr], , that loses H under forcing conditions to form [Ir(IPr)(ZnMe)H][BAr], . Crystallization of also results in the formation of its noncyclometalated isomer, [Ir(IPr)(ZnMe)][BAr], , in the solid state.

Using 2D-IR Spectroscopy to Measure the Structure, Dynamics, and Intermolecular Interactions of Proteins in HO.

Infrared (IR) spectroscopy probes molecular structure at the level of the chemical bond or functional group. In the case of proteins, the most informative band in the IR spectrum is the amide I band, which arises predominantly from the C═O stretching vibration of the peptide link. The folding of proteins into secondary and tertiary structures leads to vibrational coupling between peptide units, generating specific amide I spectral signatures that provide a fingerprint of the macromolecular conformation.

Understanding the [NiFe] Hydrogenase Active Site Environment through Ultrafast Infrared and 2D-IR Spectroscopy of the Subsite Analogue K[CpFe(CO)(CN)] in Polar and Protic Solvents.

The [CpFe(CO)(CN)] unit is an excellent structural model for the Fe(CO)(CN) moiety of the active site found in [NiFe] hydrogenases. Ultrafast infrared (IR) pump-probe and 2D-IR spectroscopy have been used to study K[CpFe(CO)(CN)] () in a range of protic and polar solvents and as a dry film. Measurements of anharmonicity, intermode vibrational coupling strength, vibrational relaxation time, and solvation dynamics of the CO and CN stretching modes of in HO, DO, methanol, dimethyl sulfoxide, and acetonitrile reveal that H-bonding to the CN ligands plays an important role in defining the spectroscopic characteristics and relaxation dynamics of the Fe(CO)(CN) unit.

Biomolecular infrared spectroscopy: making time for dynamics.

Time resolved infrared spectroscopy of biological molecules has provided a wealth of information relating to structural dynamics, conformational changes, solvation and intermolecular interactions. Challenges still exist however arising from the wide range of timescales over which biological processes occur, stretching from picoseconds to minutes or hours. Experimental methods are often limited by vibrational lifetimes of probe groups, which are typically on the order of picoseconds, while measuring an evolving system continuously over some 18 orders of magnitude in time presents a raft of technological hurdles.

Optical Screening and Classification of Drug Binding to Proteins in Human Blood Serum.

Protein-drug interactions in the human bloodstream are important factors in applications ranging from drug design, where protein binding influences efficacy and dose delivery, to biomedical diagnostics, where rapid, quantitative measurements could guide optimized treatment regimes. Current measurement approaches use multistep assays, which probe the protein-bound drug fraction indirectly and do not provide fundamental structural or dynamic information about the protein-drug interaction. We demonstrate that ultrafast 2D-IR spectroscopy can overcome these issues by providing a direct, label-free optical measurement of protein-drug binding in blood serum samples.

Tuning of B photochemistry in the CarH photoreceptor to avoid radical photoproducts.

Time-resolved infrared spectroscopy reveals the flow of electron density through coenzyme B in the light-activated, bacterial transcriptional regulator, CarH. The protein stabilises a series of charge transfer states that result in a photoresponse that avoids reactive, and potentially damaging, radical photoproducts.

Temperature-Induced Effects on the Structure of Gramicidin S.

We report on the structure of Gramicidin S (GS) in a model membrane mimetic environment represented by the amphipathic solvent 1-octanol using one-dimensional (1D) and two-dimensional (2D) IR spectroscopy. To explore potential structural changes of GS, we also performed a series of spectroscopic measurements at differing temperatures. By analyzing the amide I band and using 2D-IR spectral changes, results could be associated to the disruption of aggregates/oligomers, as well as structural and conformational changes happening in the concentrated solution of GS.

2D-IR spectroscopy of proteins in HO-A Perspective.

The form of the amide I infrared absorption band provides a sensitive probe of the secondary structure and dynamics of proteins in the solution phase. However, the frequency coincidence of the amide I band with the bending vibrational mode of HO has necessitated the widespread use of deuterated solvents. Recently, it has been demonstrated that ultrafast 2D-IR spectroscopy allows the detection of the protein amide I band in HO-based fluids, meaning that IR methods can now be applied to study proteins in physiologically relevant solvents.

Metasurface-enhanced mid-infrared spectroscopy in the liquid phase.

Vibrational spectroscopy is an important tool in chemical and biological analysis. A key issue when applying vibrational spectroscopy to dilute liquid samples is the inherently low sensitivity caused by short interaction lengths and small extinction coefficients, combined with low target molecule concentrations. Here, we introduce a novel type of surface-enhanced infrared absorption spectroscopy based on the resonance of a dielectric metasurface.

Measuring proteins in HO using 2D-IR spectroscopy: pre-processing steps and applications toward a protein library.

The ability of two-dimensional infrared (2D-IR) spectroscopy to measure the amide I band of proteins in HO rather than DO-based solvents by evading the interfering water signals has enabled in vivo studies of proteins under physiological conditions and in biofluids. Future exploitation of 2D-IR in analytical settings, from diagnostics to protein screening, will, however, require comparisons between multiple datasets, necessitating control of data collection protocols to minimize measurement-to-measurement inconsistencies. Inspired by analytical spectroscopy applications in other disciplines, we describe a workflow for pre-processing 2D-IR data that aims to simplify spectral cross-comparisons.

Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen.

NAD-reducing [NiFe] hydrogenases are valuable biocatalysts for H-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O and elevated temperatures, the soluble NAD-reducing [NiFe] hydrogenase from (SH) is O-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications.

Detection of paracetamol binding to albumin in blood serum using 2D-IR spectroscopy.

Binding of drugs to blood serum proteins can influence both therapeutic efficacy and toxicity. The ability to measure the concentrations of protein-bound drug molecules quickly and with limited sample preparation could therefore have considerable benefits in biomedical and pharmaceutical applications. Vibrational spectroscopies provide data quickly but are hampered by complex, overlapping protein amide I band profiles and water absorption.

Combining steady state and temperature jump IR spectroscopy to investigate the allosteric effects of ligand binding to dsDNA.

Changes in the structural dynamics of double stranded (ds)DNA upon ligand binding have been linked to the mechanism of allostery without conformational change, but direct experimental evidence remains elusive. To address this, a combination of steady state infrared (IR) absorption spectroscopy and ultrafast temperature jump IR absorption measurements has been used to quantify the extent of fast (∼100 ns) fluctuations in (ds)DNA·Hoechst 33258 complexes at a range of temperatures. Exploiting the direct link between vibrational band intensities and base stacking shows that the absolute magnitude of the change in absorbance caused by fast structural fluctuations following the temperature jump is only weakly dependent on the starting temperature of the sample.

Detection of Glycine as a Model Protein in Blood Serum Using 2D-IR Spectroscopy.

Glycine (Gly) is used as a model system to evaluate the ability of ultrafast two-dimensional infrared (2D-IR) spectroscopy to detect and quantify the low-molecular-weight proteinaceous components of blood serum. Combining data acquisition schemes to suppress absorption bands of HO that overlap with the protein amide I band with analysis of peak patterns appearing in the off-diagonal region of the 2D-IR spectrum allows separation of the Gly spectral signature from that of the dominant protein fraction of serum in a transmission-mode 2D-IR measurement without any sample manipulation, e.g.

Experimental observation of nanophase segregation in aqueous salt solutions around the predicted liquid-liquid transition in water.

The liquid-liquid transition in supercooled liquid water, predicted to occur around 220 K, is controversial due to the difficulty of studying it caused by competition from ice crystallization (the so-called "no man's land"). In aqueous solutions, it has been predicted to give rise to phase separation on a nanometer scale between a solute-rich high-density phase and a water-rich low-density phase. Here we report direct experimental evidence for the formation of a nanosegregated phase in eutectic aqueous solutions of LiCl and LiSCN where the presence of crystalline water can be experimentally excluded.

Time-Resolved Temperature-Jump Infrared Spectroscopy at a High Repetition Rate.

Time-resolved temperature-jump infrared absorption spectroscopy at a 0.5 to 1 kHz repetition rate is presented. A 1 kHz neodymium-doped yttrium aluminum garnet (Nd:YAG) laser pumping an optical parametric oscillator provided >70 µJ, 3.

Two-dimensional infrared spectroscopy: an emerging analytical tool?

Ultrafast two-dimensional infrared (2D-IR) spectroscopy has provided valuable insights into biomolecular structure and dynamics, but recent progress in laser technology and data analysis methods have demonstrated the potential for high throughput 2D-IR measurements and analytical applications. Using 2D-IR as an analytical tool requires a different approach to data collection and analysis compared to pure research applications however and, in this review, we highlight progress towards usage of 2D-IR spectroscopy in areas relevant to biomedical, pharmaceutical and analytical molecular science. We summarise the technical and methodological advances made to date and discuss the challenges that still face 2D-IR spectroscopy as it attempts to transition from the state-of-the-art laser laboratory to the standard suite of analytical tools.

2D-Infrared Spectroscopy of Proteins in Water: Using the Solvent Thermal Response as an Internal Standard.

Ultrafast two-dimensional infrared (2D-IR) spectra can now be obtained in a matter of seconds, opening up the possibility of high-throughput screening applications of relevance to the biomedical and pharmaceutical sectors. Determining quantitative information from 2D-IR spectra recorded on different samples and different instruments is however made difficult by variations in beam alignment, laser intensity, and sample conditions. Recently, we demonstrated that 2D-IR spectroscopy of the protein amide I band can be performed in aqueous (HO) rather than deuterated (DO) solvents, and we now report a method that uses the magnitude of the associated thermal response of HO as an internal normalization standard for 2D-IR spectra.

Understanding the structure and dynamics of hydrogenases by ultrafast and two-dimensional infrared spectroscopy.

Hydrogenases are valuable model enzymes for sustainable energy conversion approaches using H, but rational utilization of these base-metal biocatalysts requires a detailed understanding of the structure and dynamics of their complex active sites. The intrinsic CO and CN ligands of these metalloenzymes represent ideal chromophores for infrared (IR) spectroscopy, but structural and dynamic insight from conventional IR absorption experiments is limited. Here, we apply ultrafast and two-dimensional (2D) IR spectroscopic techniques, for the first time, to study hydrogenases in detail.

Uncovering the Early Stages of Domain Melting in Calmodulin with Ultrafast Temperature-Jump Infrared Spectroscopy.

The signaling protein calmodulin (CaM) undergoes a well-known change in secondary structure upon binding Ca, but the structural plasticity of the Ca-free state is linked to CaM functionality. Variable temperature studies of -CaM indicate two structural transitions at 46 and 58 °C that are assigned to melting of the C- and N-terminal domains, respectively, but the molecular mechanism of domain unfolding is unknown. We report temperature-jump time-resolved infrared (IR) spectroscopy experiments designed to target the first steps in the C-terminal domain melting transition of human -CaM.

Measuring proteins in HO with 2D-IR spectroscopy.

The amide I infrared band of proteins is highly sensitive to secondary structure, but studies under physiological conditions are prevented by strong, overlapping water absorptions, motivating the widespread use of deuterated solutions. H/D exchange raises fundamental questions regarding the impact of increased mass on protein dynamics, while deuteration is impractical for biomedical or commercial applications of protein IR spectroscopy. We show that 2D-IR spectroscopy can avoid this problem because the 2D-IR amide I signature of proteins dominates that of water even at sub-millimolar protein concentrations.