Using 2D-IR Spectroscopy to Measure the Structure, Dynamics, and Intermolecular Interactions of Proteins in H2O.

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. Ultrafast two-dimensional IR (2D-IR) spectroscopy allows the amide I band of a protein to be spread over a second frequency dimension in a way that mirrors 2D-NMR methods. This means that amide I 2D-IR spectroscopy produces a spectral map that is exquisitely sensitive to protein structure and dynamics and so provides detailed insights that cannot be matched by IR absorption spectroscopy. As a result, 2D-IR spectroscopy has emerged as a powerful tool for probing protein structure and dynamics over a broad range of time and length scales in the solution phase at room temperature. However, the protein amide I band coincides with an IR absorption from the bending vibration of water (δHOH), the natural biological solvent. To circumvent this problem, protein IR studies are routinely performed in D2O solutions because H/D substitution shifts the solvent bending mode (δDOD) to a lower frequency, revealing the amide I band. While effective, this method raises fundamental questions regarding the impact of the change in solvent mass on the structural or solvation dynamics of the protein and the removal of the energetic resonance between solvent and solute.In this Account, a series of studies applying 2D-IR to study the spectroscopy and dynamics of proteins in H2O-rich solvents is reviewed. A comparison of IR absorption spectroscopy and 2D-IR spectroscopy of protein-containing fluids is used to demonstrate the basis of the approach before a series of applications is presented. These range from measurements of fundamental protein biophysics to recent applications of machine learning to gain insight into protein-drug binding in complex mixtures. An outlook is presented, considering the potential for 2D-IR measurements to contribute to our understanding of protein behavior under near-physiological conditions, along with an evaluation of the obstacles that still need to be overcome.

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 in vivo 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. Four commonly prescribed drugs, known to bind to human serum albumin (HSA), were added to pooled human serum at physiologically relevant concentrations. In each case, spectral changes to the amide I band of the serum sample were observed, consistent with binding to HSA, but were distinct for each of the four drugs. A machine-learning-based classification of the serum samples achieved a total cross-validation prediction accuracy of 92% when differentiating serum-only samples from those with a drug present. Identification on a per-drug basis achieved correct drug identification in 75% of cases. These unique spectroscopic signatures of the drug-protein interaction thus enable the detection and differentiation of drug containing samples and give structural insight into the binding process as well as quantitative information on protein-drug binding. Using currently available instrumentation, the 2D-IR data acquisition required just 1 min and 10 µL of serum per sample, and so these results pave the way to fast, specific, and quantitative measurements of protein-drug binding in vivo with potentially invaluable applications for the development of novel therapies and personalized medicine.

2D-IR spectroscopy of proteins in H2O-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 H2O 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 H2O-based fluids, meaning that IR methods can now be applied to study proteins in physiologically relevant solvents. In this perspective, we describe the basis of the 2D-IR method for observing the protein amide I band in H2O and show how this development has the potential to impact areas ranging from our fundamental appreciation of protein structural dynamics to new applications for 2D-IR spectroscopy in the analytical and biomedical sciences. In addition, we discuss how the spectral response of water, rather than being a hindrance, now provides a basis for new approaches to data pre-processing, standardization of 2D-IR data collection, and signal quantification. Ultimately, we visualize a direction of travel toward the creation of 2D-IR spectral libraries that can be linked to advanced computational methods for use in high-throughput protein screening and disease diagnosis.

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 H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temperatures, the soluble NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.

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. Here, we show that two-dimensional infrared (2D-IR) spectroscopy can achieve rapid detection and quantification of paracetamol binding to serum albumin in blood serum at physiologically-relevant levels with no additional sample processing. By measuring changes to the amide I band of serum albumin caused by structural and dynamic impacts of paracetamol binding we show that drug concentrations as low as 7 µM can be detected and that the availability of albumin for paracetamol binding is less than 20% in serum samples, allowing identification of paracetamol levels consistent with a patient overdose.

Ultrafast 2D-IR spectroscopy of [NiFe] hydrogenase from E. coli reveals the role of the protein scaffold in controlling the active site environment.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of Escherichia coli Hyd-1 (EcHyd-1) reveals the structural and dynamic influence of the protein scaffold on the Fe(CO)(CN)2 unit of the active site. Measurements on as-isolated EcHyd-1 probed a mixture of active site states including two, which we assign to Nir-SI/II, that have not been previously observed in the E. coli enzyme. Explicit assignment of carbonyl (CO) and cyanide (CN) stretching bands to each state is enabled by 2D-IR. Energies of vibrational levels up to and including two-quantum vibrationally excited states of the CO and CN modes have been determined along with the associated vibrational relaxation dynamics. The carbonyl stretching mode potential is well described by a Morse function and couples weakly to the cyanide stretching vibrations. In contrast, the two CN stretching modes exhibit extremely strong coupling, leading to the observation of formally forbidden vibrational transitions in the 2D-IR spectra. We show that the vibrational relaxation times and structural dynamics of the CO and CN ligand stretching modes of the enzyme active site differ markedly from those of a model compound K[CpFe(CO)(CN)2] in aqueous solution and conclude that the protein scaffold creates a unique biomolecular environment for the NiFe site that cannot be represented by analogy to simple models of solvation.

Measuring proteins in H2O 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 H2O rather than D2O-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. Our approach exploits the thermal water signal that is collected simultaneously with, but is temporally separated from the amide I response to guide custom baseline correction and spectral normalization strategies before combining them with Principal Component noise reduction tools. Case studies show that application of elements of the pre-processing workflow to previously published data enables improvements in quantification accuracy and detection limits. We subsequently apply the complete workflow in a new pilot study, testing the ability of a prototype library of 2D-IR spectra to quantify the four major protein constituents of blood serum in a single, label-free measurement. These advances show progress toward the robust data handling strategies that will be necessary for future applications of 2D-IR to pharmaceutical or biomedical problems.

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 H2O 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., filtration or drying. 2D-IR spectra of blood serum samples supplemented with varying concentrations of Gly were obtained, and a range of data analysis methods compared, leading to a detection limit of ∼3 mg/mL for Gly. The reported methodology provides a platform for a critical assessment of the sensitivity of 2D-IR for measuring the concentrations of amino acids, peptides, and low-molecular-weight proteins present in serum samples. We conclude that, in the case of several clinically relevant diagnostic molecules and their combinations, the potential exists for 2D-IR to complement IR absorption methods as the benefits of the second frequency dimension offered by 2D-IR spectroscopy outweigh the added technical complexity of the measurement.

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. The observed fast dynamics are some two orders of magnitude faster than strand separation and associated with all points along the 10-base pair duplex d(GCATATATCC). Binding the Hoechst 33258 ligand causes a small but consistent reduction in the extent of these fast fluctuations of base pairs located outside of the ligand binding region. These observations point to a ligand-induced reduction in the flexibility of the dsDNA near the binding site, consistent with an estimated allosteric propagation length of 15 Å, about 5 base pairs, which agrees well with both molecular simulation and coarse-grained statistical mechanics models of allostery leading to cooperative ligand binding.

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 (H2O) rather than deuterated (D2O) solvents, and we now report a method that uses the magnitude of the associated thermal response of H2O as an internal normalization standard for 2D-IR spectra. Using the water response, which is temporally separated from the protein signal, to normalize the spectra allows significant reduction of the impact of measurement-to-measurement fluctuations on the data. We demonstrate that this normalization method enables creation of calibration curves for measurement of absolute protein concentrations and facilitates reproducible difference spectroscopy methodologies. These advances make significant progress toward the robust data handling strategies that will be essential for the realization of automated spectral analysis tools for large scale 2D-IR screening studies of protein-containing solutions and biofluids.

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.

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. Femtosecond infrared and Raman spectroscopies are used to determine the temperature-dependent structuring of water, the solvation of the SCN- anion, and the size of the phase segregated domains.

Tunable Supramolecular Gel Properties by Varying Thermal History.

The possibility of using differential pre-heating prior to supramolecular gelation to control the balance between hydrogen-bonding and aromatic stacking interactions in supramolecular gels and obtain consequent systematic regulation of structure and properties is demonstrated. Using a model aromatic peptide amphiphile, Fmoc-tyrosyl-leucine (Fmoc-YL) and a combination of fluorescence, infrared, circular dichroism and NMR spectroscopy, it is shown that the balance of these interactions can be adjusted by temporary exposure to elevated temperatures in the range 313-365 K, followed by supramolecular locking in the gel state by cooling to room temperature. Distinct regimes can be identified regarding the balance between H-bonding and aromatic stacking interactions, with a transition point at 333 K. Consequently, gels can be obtained with customizable properties, including supramolecular chirality and gel stiffness. The differential supramolecular structures also result in changes in proteolytic stability, highlighting the possibility of obtaining a range of supramolecular architectures from a single molecular structure by simply controlling the pre-assembly temperature.

Rapid Screening of DNA-Ligand Complexes via 2D-IR Spectroscopy and ANOVA-PCA.

Two-dimensional infrared spectroscopy (2D-IR) is well established as a specialized, high-end technique for measuring structural and solvation dynamics of biological molecules. Recent technological developments now make it possible to acquire time-resolved 2D-IR spectra within seconds, and this opens up the possibility of screening-type applications comparing spectra spanning multiple samples. However, such applications bring new challenges associated with finding accurate, efficient methodologies to analyze large data sets in a timely, informative manner. Here, we demonstrate such an application by screening 2016 2D-IR spectra of 12 double-stranded DNA oligonucleotides obtained in the presence and absence of binding therapeutic molecule Hoechst 33258. By applying analysis of variance combined with principal component analysis (ANOVA-PCA) to 2D-IR data for the first time, we demonstrate the ability to efficiently retrieve the base composition of a DNA sequence and discriminate ligand-DNA complexes from unbound sequences. We further show accurate differentiation of the induced-fit and rigid-body binding modes that is key to identifying optimal binding interactions of Hoechst 33258, while ANOVA-PCA results across the full sequence range correlate directly with thermodynamic indicators of ligand-binding strength that require significantly longer data acquisition times to obtain.

Quantifying Secondary Structure Changes in Calmodulin Using 2D-IR Spectroscopy.

Revealing the details of biomolecular processes in solution needs tools that can monitor structural dynamics over a range of time and length scales. We assess the ability of 2D-IR spectroscopy in combination with multivariate data analysis to quantify changes in secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM) as a function of temperature and Ca2+ concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca2+-free (apo) CaM (reduction in α-helix structure by 13% (CD) and 15% (2D)). 2D-IR also allows accurate differentiation between melting transitions and generic heating effects observed in the more thermally stable Ca2+-bound (holo) CaM. The functionally relevant random-coil-α-helix transition associated with Ca2+ uptake that involves just 7-8 out of a total of 148 amino acid residues was clearly detected. Temperature-dependent Molecular Dynamics (MD) simulations show that apo-CaM exists in dynamic equilibrium with holo-like conformations, while Ca2+ uptake reduces conformational flexibility. The ability to combine quantitative structural insight from 2D-IR with MD simulations thus offers a powerful approach for measuring subtle protein conformational changes in solution.

Computing infrared spectra of proteins using the exciton model.

The ability to compute from first principles the infrared spectrum of a protein in solution phase representing a biological system would provide a useful connection to atomistic models of protein structure and dynamics. Indeed, such calculations are a vital complement to 2DIR experimental measurements, allowing the observed signals to be interpreted in terms of detailed structural and dynamical information. In this article, we have studied nine structurally and spectroscopically well-characterized proteins, representing a range of structural types. We have simulated the equilibrium conformational dynamics in an explicit point charge water model. Using the resulting trajectories based on MD simulations, we have computed the one and two dimensional infrared spectra in the Amide I region, using an exciton approach, in which a local mode basis of carbonyl stretches is considered. The role of solvent in shifting the Amide I band (by 30 to 50 cm-1 ) is clearly evident. Similarly, the conformational dynamics contribute to the broadening of peaks in the spectrum. The inhomogeneous broadening in both the 1D and 2D spectra reflects the significant conformational diversity observed in the simulations. Through the computed 2D cross-peak spectra, we show how different pulse schemes can provide additional information on the coupled vibrations. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Ultrafast 2D-IR and optical Kerr effect spectroscopy reveal the impact of duplex melting on the structural dynamics of DNA.

Changes in the structural and solvation dynamics of a 15mer AT DNA duplex upon melting of the double-helix are observed by a combination of ultrafast two-dimensional infrared (2D-IR) and optical Kerr-effect (OKE) spectroscopies. 2D-IR spectroscopy of the vibrational modes of the DNA bases reveal signature off-diagonal peaks arising from coupling and energy transfer across Watson-Crick paired bases that are unique to double-stranded DNA (ds-DNA). Spectral diffusion of specific base vibrational modes report on the structural dynamics of the duplex and the minor groove, which is predicted to contain a spine of hydration. Changes in these dynamics upon melting are assigned to increases in the degree of mobile solvent access to the bases in single-stranded DNA (ss-DNA) relative to the duplex. OKE spectra exhibit peaks that are assigned to specific long-range phonon modes of ds- and ss-DNA. Temperature-related changes in these features correlate well with those obtained from the 2D-IR spectra although the melting temperature of the ds-DNA phonon band is slightly higher than that for the Watson-Crick modes, suggesting that a degree of long-range duplex structure survives the loss of Watson-Crick hydrogen bonding. These results demonstrate that the melting of ds-DNA disrupts helix-specific structural dynamics encompassing length scales ranging from mode delocalisation in the Watson-Crick base pairs to long-range phonon modes that extend over multiple base pairs and which may play a role in molecular recognition of DNA.

Combining random forest and 2D correlation analysis to identify serum spectral signatures for neuro-oncology.

Fourier transform infrared (FTIR) spectroscopy has long been established as an analytical technique for the measurement of vibrational modes of molecular systems. More recently, FTIR has been used for the analysis of biofluids with the aim of becoming a tool to aid diagnosis. For the clinician, this represents a convenient, fast, non-subjective option for the study of biofluids and the diagnosis of disease states. The patient also benefits from this method, as the procedure for the collection of serum is much less invasive and stressful than traditional biopsy. This is especially true of patients in whom brain cancer is suspected. A brain biopsy is very unpleasant for the patient, potentially dangerous and can occasionally be inconclusive. We therefore present a method for the diagnosis of brain cancer from serum samples using FTIR and machine learning techniques. The scope of the study involved 433 patients from whom were collected 9 spectra each in the range 600-4000 cm(-1). To begin the development of the novel method, various pre-processing steps were investigated and ranked in terms of final accuracy of the diagnosis. Random forest machine learning was utilised as a classifier to separate patients into cancer or non-cancer categories based upon the intensities of wavenumbers present in their spectra. Generalised 2D correlational analysis was then employed to further augment the machine learning, and also to establish spectral features important for the distinction between cancer and non-cancer serum samples. Using these methods, sensitivities of up to 92.8% and specificities of up to 91.5% were possible. Furthermore, ratiometrics were also investigated in order to establish any correlations present in the dataset. We show a rapid, computationally light, accurate, statistically robust methodology for the identification of spectral features present in differing disease states. With current advances in IR technology, such as the development of rapid discrete frequency collection, this approach is of importance to enable future clinical translation and enables IR to achieve its potential.

Detection of Transient Intermediates Generated from Subsite Analogues of [FeFe] Hydrogenases.

This article reviews the application of transient techniques in the elucidation of electron, proton, and photon chemistry related to the catalytic subsite of [FeFe] hydrogenase from the perspective of research in this area carried out at the UEA and Strathclyde laboratories. The detection of mixed-valence states, bridging CO intermediates, paramagnetic hydrides, and coordinatively unsaturated species has both informed understanding of biological catalysis and stimulated the search for stable analogues of key structural motifs likely involved in turnover states.

Multidimensional infrared spectroscopy reveals the vibrational and solvation dynamics of isoniazid.

The results of infrared spectroscopic investigations into the band assignments, vibrational relaxation, and solvation dynamics of the common anti-tuberculosis treatment Isoniazid (INH) are reported. INH is known to inhibit InhA, a 2-trans-enoyl-acyl carrier protein reductase enzyme responsible for the maintenance of cell walls in Mycobacterium tuberculosis but as new drug-resistant strains of the bacterium appear, next-generation therapeutics will be essential to combat the rise of the disease. Small molecules such as INH offer the potential for use as a biomolecular marker through which ultrafast multidimensional spectroscopies can probe drug binding and so inform design strategies but a complete characterization of the spectroscopy and dynamics of INH in solution is required to inform such activity. Infrared absorption spectroscopy, in combination with density functional theory calculations, is used to assign the vibrational modes of INH in the 1400-1700 cm(-1) region of the infrared spectrum while ultrafast multidimensional spectroscopy measurements determine the vibrational relaxation dynamics and the effects of solvation via spectral diffusion of the carbonyl stretching vibrational mode. These results are discussed in the context of previous linear spectroscopy studies on solid-phase INH and its usefulness as a biomolecular probe.