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 (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.

Measuring proteins in H2O 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. 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 clinically-relevant 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.

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases.

The role of specific cleavage of transcription repressor proteins by proteases and how this may be related to the emerging theme of dinucleotides as cellular signaling molecules is poorly characterized. The transcription repressor NmrA of Aspergillus nidulans discriminates between oxidized and reduced dinucleotides, however, dinucleotide binding has no effect on its interaction with the zinc finger in the transcription activator AreA. Protease activity in A. nidulans was assayed using NmrA as the substrate, and was absent in mycelium grown under nitrogen sufficient conditions but abundant in mycelium starved of nitrogen. One of the proteases was purified and identified as the protein Q5BAR4 encoded by the gene AN2366.2. Fluorescence confocal microscopy showed that the nuclear levels of NmrA were reduced approximately 38% when mycelium was grown on nitrate compared to ammonium and absent when starved of nitrogen. Proteolysis of NmrA occurred in an ordered manner by preferential digestion within a C-terminal surface exposed loop and subsequent digestion at other sites. NmrA digested at the C-terminal site was unable to bind to the AreA zinc finger. These data reveal a potential new layer of control of nitrogen metabolite repression by the ordered proteolytic cleavage of NmrA. NmrA digested at the C-terminal site retained the ability to bind NAD(+) and showed a resistance to further digestion that was enhanced by the presence of NAD(+). This is the first time that an effect of dinucleotide binding to NmrA has been demonstrated.