2D-IR spectroscopy is used to measure protein amide I bands in water, avoiding the need for deuteration. We show that H/D exchange affects protein vibrational relaxation dynamics and that the ability to perform 2D-IR in water enables blood serum protein analysis.

In recent years, the application of vibrational spectroscopy in biomedical research has rapidly expanded; covering aspects of pharmaceutical development, to point-of-care technologies. Vibrational spectroscopy techniques such as Fourier-transform IR (FTIR), and Raman spectroscopy have been at the forefront of this movement, with their complementary information able to shine light onto a range of medical applications. As a relative newcomer to biomedical applications, two-dimensional (2D)-IR is also gaining traction in the field. Here we describe the recent development of these techniques as analytical tools in medical science, and their relative advancements towards the clinic.

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. Using equine blood serum as a test system, we investigate the significant implications of being able to measure the spectroscopy and dynamics of proteins in water, demonstrating relevance in areas ranging from fundamental science to the clinic. Measurements of vibrational relaxation dynamics of serum proteins reveals that deuteration slows down the rate of amide I vibrational relaxation by >10%, indicating a dynamic impact of isotopic exchange in some proteins. The unique link between protein secondary structure and 2D-IR amide I lineshape allows differentiation of signals due to albumin and globulin protein fractions in serum leading to measurements of the biomedically-important albumin to globulin ratio (AGR) with an accuracy of 4% across a clinicallyrelevant range. Furthermore, we demonstrate that 2D-IR spectroscopy enables differentiation of the structurally similar globulin proteins IgG, IgA and IgM, opening up a straightforward spectroscopic approach to measuring levels of serum proteins that are currently only accessible via biomedical laboratory testing.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy is a powerful probe of the structural and vibrational dynamics of proteins and enzymes in the solution phase. Until recently, relatively few applications of 2D-IR to DNA had been reported, but this is beginning to change rapidly, showing that the vibrational modes of DNA are sensitive reporters of base pairing and stacking and allowing site-specific probing of the nature of the complex interactions of the DNA macromolecule with its solvent environment. Most recently, 2D-IR spectroscopy has been used to probe the minor-groove ligand binding mechanism and reveals the melting of double-stranded DNA in real time, offering the potential for 2D-IR to provide mechanistic insight into the behavior of this most fundamental of biological molecules in the solution phase. The experimental methods used to obtain 2D-IR spectra are first described along with a discussion of the 2D-IR spectral features relevant to DNA studies before a review of the current state of the art of 2D-IR spectroscopy applications to DNA is presented.

The effect of oligomer length on the vibrational mode coupling and energy relaxation mechanisms of AT-rich DNA oligomers in double- and single-stranded conformations has been investigated using two-dimensional infrared spectroscopy. Vibrational coupling of modes of the DNA bases to the symmetric stretching vibration of the backbone phosphate group was observed for oligomers long enough to form duplex-DNA structures. The coupling was lost upon melting of the duplex. No significant effect of oligomer length or DNA secondary structure was found on either the timescale for vibrational relaxation of the base modes or the mechanism, which was consistent with a cascade process from base modes to intermediate modes, some of which are located on the deoxyribose group, and subsequently to the phosphate backbone. The study shows that vibrational coupling between base and backbone requires formation of the double-helix structure while vibrational energy management is an inherent property of the nucleotide.

The induced fit binding model describes a conformational change occurring when a small molecule binds to its biomacromolecular target. The result is enhanced noncovalent interactions between the ligand and biomolecule. Induced fit is well-established for small molecule-protein interactions, but its relevance to small molecule-DNA binding is less clear. We investigate the molecular determinants of Hoechst33258 binding to its preferred A-tract sequence relative to a suboptimal alternating A-T sequence. Results from two-dimensional infrared spectroscopy, which is sensitive to H-bonding and molecular structure changes, show that Hoechst33258 binding results in loss of the minor groove spine of hydration in both sequences, but an additional perturbation of the base propeller twists occurs in the A-tract binding region. This induced fit maximizes favorable ligand-DNA enthalpic contributions in the optimal binding case and demonstrates that controlling the molecular details that induce subtle changes in DNA structure may hold the key to designing next-generation DNA-binding molecules.

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.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of a 15-mer A-T DNA duplex in solution has revealed structure-dependent vibrational coupling and energy transfer processes linking bases with the sugar phosphate backbone. Duplex melting induces significant changes in the positions of off diagonal peaks linking carbonyl and ring-stretching vibrational modes of the adenine and thymine bases with vibrations of the phosphate group and phosphodiester linkage. These indicate that Watson Crick hydrogen bonding and helix formation lead to a unique vibrational coupling arrangement of base vibrational modes with those of the phosphate unit. On the basis of observations from time-resolved 2D-IR data, we conclude that rapid energy transfer processes;occur between base and backbone, mediated by additional modes located on the deoxyribose moiety within the same nucleotide. These relaxation dynamics are insensitive to duplex melting, showing that efficient intramolecular energy relaxation to the solvent via the phosphate groups is the key to excess energy dissipation in both single- and double-stranded DNA.

Ultrafast, multi-dimensional infrared spectroscopy, in the form of 2D-IR and pump-probe measurements, has been employed to investigate the effect of substrate binding on the structural dynamics of the horseradish peroxidase (HRP) enzyme. Using nitric oxide bound to the ferric haem of HRP as a sensitive probe of local dynamics, we report measurements of the frequency fluctuations (spectral diffusion) and vibrational lifetime of the NO stretching mode with benzohydroxamic acid (BHA) located in the substrate-binding position at the periphery of the haem pocket, in both D2O and H2O solvents. The results reveal that, with BHA bound to the enzyme, the local structural dynamics are insensitive to H/D exchange. These results are in stark contrast to those found in studies of the substrate-free enzyme, which demonstrated that the local chemical and dynamic environment of the haem ligand is influenced by water molecules. In light of the large changes in solvent accessibility caused by substrate binding, we discuss the potential for varying roles for the solvent in the haem pocket of HRP at different stages along the reaction coordinate of the enzymatic mechanism.