Structure Changes of a Membrane Polypeptide under an Applied Voltage Observed with Surface-Enhanced 2D IR Spectroscopy.

The structures of many membrane-bound proteins and polypeptides depend on the membrane potential. However, spectroscopically studying their structures under an applied field is challenging, because a potential is difficult to generate across more than a few bilayers. We study the voltage-dependent structures of the membrane-bound polypeptide, alamethicin, using a spectroelectrochemical cell coated with a rough, gold film to create surface plasmons. The plasmons sufficiently enhance the 2D IR signal to measure a single bilayer. The film is also thick enough to conduct current and thereby apply a potential. The 2D IR spectra resolve features from both 310- and α-helical structures and cross-peaks connecting the two. We observe changes in the peak intensity, not their frequencies, upon applying a voltage. A similar change occurs with pH, which is known to alter the angle of alamethicin relative to the surface normal. The spectra are modeled using a vibrational exciton Hamiltonian, and the voltage-dependent spectra are consistent with a change in angle of the 310- and α-helices in the membrane from 55 to 44°and from 31 to 60°, respectively. The 310- and α-helices are coupled by approximately 10 cm-1. These experiments provide new structural information about alamethicin under a potential difference and demonstrate a technique that might be applied to voltage-gated membrane proteins and compared to molecular dynamics structures.

A Proposed Method to Obtain Surface Specificity with Pump-Probe and 2D Spectroscopies.

Surfaces and interfaces are ubiquitous in nature. From cell membranes, to photovoltaic thin films, surfaces have important function in both biological and materials systems. Spectroscopic techniques have been developed to probe systems like these, such as sum frequency generation (SFG) spectroscopies. The advantage of SFG spectroscopy, a second-order spectroscopy, is that it can distinguish between signals produced from molecules in the bulk versus on the surface. We propose a polarization scheme for third-order spectroscopy experiments, such as pump-probe and 2D spectroscopy, to select for surface signals and not bulk signals. This proposed polarization condition uses one pulse perpendicular compared to the other three to isolate cross-peaks arising from molecules with polar and uniaxial (i.e., biaxial) order at a surface, while removing the signal from bulk isotropic molecules. In this work, we focus on two of these cases: XXXY and YYYX, which differ by the sign of the cross-peak they create. We compare this technique to SFG spectroscopy and vibrational circular dichroism to provide insight to the behavior of the cross-peak signal. We propose that these singularly cross-polarized schemes provide odd-ordered spectroscopies the surface-specificity typically associated with even-ordered techniques.

Monolayer Sensitivity Enables a 2D IR Spectroscopic Immuno-biosensor for Studying Protein Structures: Application to Amyloid Polymorphs.

Immunosensors use antibodies to detect and quantify biomarkers of disease, though the sensors often lack structural information. We create a surface-sensitive two-dimensional infrared (2D IR) spectroscopic immunosensor for studying protein structures. We tether antibodies to a plasmonic surface, flow over a solution of amyloid proteins, and measure the 2D IR spectra. The 2D IR spectra provide a global assessment of antigen structure, and isotopically labeled proteins give residue-specific structural information. We report the 2D IR spectra of fibrils and monomers using a polyclonal antibody that targets human islet amyloid polypeptide (hIAPP). We observe two fibrillar polymorphs differing in their structure at the G24 residue, which supports the hypothesis that hIAPP polymorphs form from a common oligomeric intermediate. This work provides insight into the structure of hIAPP, establishes a new method for studying protein structures using 2D IR spectroscopy, and creates a spectroscopic immunoassay applicable for studying a wide range of biomarkers.

Amyloid found in human cataracts with two-dimensional infrared spectroscopy.

UV light and other factors damage crystallin proteins in the eye lens, resulting in cataracts that scatter light and affect vision. Little information exists about protein structures within these disease-causing aggregates. We examined postmortem lens tissue from individuals with and without cataracts using 2D infrared (2DIR) spectroscopy. Amyloid ß-sheet secondary structure was detected in cataract lenses along with denatured structures. No amyloid structures were found in lenses from juveniles, but mature lenses with no cataract diagnosis also contained amyloid, indicating that amyloid structures begin forming before diagnosis. Light scatters more strongly in regions with amyloid structure, and UV light induces amyloid ß-sheet structures, linking the presence of amyloid structures to disease pathology. Establishing that age-related cataracts involve amyloid structures gives molecular insight into a common human affliction and provides a possible structural target for pharmaceuticals as an alternative to surgery.

Enhancing the signal strength of surface sensitive 2D IR spectroscopy.

Spectroscopic techniques that are capable of measuring surfaces and interfaces must overcome two technical challenges: one, the low coverage of molecules at the surface, and two, discerning between signals from the bulk and surface. We present surface enhanced attenuated reflection 2D infrared (SEAR 2D IR) spectroscopy, a method that combines localized surface plasmons with a reflection pump-probe geometry to achieve monolayer sensitivity. The method is demonstrated at 6 µm with the amide I band of a model peptide, a cysteine terminated α-helical peptide tethered to a gold surface. Using SEAR 2D IR spectroscopy, the signal from this sample is enhanced 20 000-times over a monolayer on a dielectric surface. Like attenuated total reflection IR spectroscopy, SEAR 2D IR spectroscopy can be applied to strongly absorbing solvents. We demonstrated this capability by solvating a peptide monolayer with H2O, which cannot normally be used when measuring the amide I band. SEAR 2D IR spectroscopy will be advantageous for studying chemical reactions at electrochemical surfaces, interfacial charge transfer in photovoltaics, and structural changes of transmembrane proteins in lipid membranes.

Invariance of Water Permeance through Size-Differentiated Graphene Oxide Laminates.

Laminates made of graphene oxide nanosheets have been shown to exhibit high water permeance and salt rejection and, therefore, have generated immense interest from the scientific community due to their potential in separation applications. However, there is no clear consensus on the water-transport pathways through such laminates. In this study, we synthesized chemically identical graphene oxide nanosheets with 2 orders of magnitude difference in lateral sizes and measured water permeance through laminates of different thicknesses fabricated by pressure-assisted deposition of these nanosheets. Our results reveal that water permeance through these laminates is nearly the same despite such massive difference in lateral sheet size. Furthermore, we simulated fluid flow through laminates using an interconnected nanochannel network model for comparison with experiments. The simulations in combination with the experimental data show that it is unlikely that the dominant fluid transport pathway is a circuitous, lateral pathway around individual sheets, as has been proposed in some studies. Rather, nonideal factors including trans-sheet flow through pinhole defects in sheet interiors and/or flow-through regions arising from imperfect stacking in the laminates can significantly affect the fluid transport pathways. The presence of such nonidealities is also supported by thickness- and time-dependent measurements of permeance and by infrared spectroscopy, which indicates that water predominantly adopts a bulk-like structure in the laminates. These analyses are significant steps toward understanding water transport through graphene oxide laminates and provide further insight toward the structure of water inside these materials, which could have immense potential in next-generation separation applications.

Spectroscopic Signature for Stable ß-Amyloid Fibrils versus ß-Sheet-Rich Oligomers.

We use two-dimensional IR (2D IR) spectroscopy to explore fibril formation for the two predominant isoforms of the ß-amyloid (Aß1-40 and Aß1-42) protein associated with Alzheimer's disease. Two-dimensional IR spectra resolve a transition at 1610 cm-1 in Aß fibrils that does not appear in other Aß aggregates, even those with predominantly ß-sheet-structure-like oligomers. This transition is not resolved in linear IR spectroscopy because it lies under the broad band centered at 1625 cm-1, which is the traditional infrared signature for amyloid fibrils. The feature is prominent in 2D IR spectra because 2D lineshapes are narrower and scale nonlinearly with transition dipole strengths. Transmission electron microscopy measurements demonstrate that the 1610 cm-1 band is a positive identification of amyloid fibrils. Sodium dodecyl sulfate micelles that solubilize and disaggregate preaggregated Aß samples deplete the 1625 cm-1 band but do not affect the 1610 cm-1 band, demonstrating that the 1610 cm-1 band is due to very stable fibrils. We demonstrate that the 1610 cm-1 transition arises from amide I modes by mutating out the only side-chain residue that could give rise to this transition, and we explore the potential structural origins of the transition by simulating 2D IR spectra based on Aß crystal structures. It was not previously possible to distinguish stable Aß fibrils from the less stable ß-sheet-rich oligomers with infrared light. This 2D IR signature will be useful for Alzheimer's research on Aß aggregation, fibril formation, and toxicity.

Not All ß-Sheets Are the Same: Amyloid Infrared Spectra, Transition Dipole Strengths, and Couplings Investigated by 2D IR Spectroscopy.

We report the transition dipole strengths and frequencies of the amyloid ß-sheet amide I mode for the aggregated proteins amyloid-ß1-40, calcitonin, α-synuclein, and glucagon. According to standard vibrational coupling models for proteins, the frequencies of canonical ß-sheets are set by their size and structural and environmental disorder, which determines the delocalization length of the vibrational excitons. The larger the delocalization the lower the frequency of the main infrared-allowed transition, A⊥. The models also predict an accompanying increase in transition dipole strength. For the proteins measured here, we find no correlation between transition dipole strengths and amyloid ß-sheet transition frequency. To understand this observation, we have extracted from the protein data bank crystal structures of amyloid peptides from which we calculate the amide I vibrational couplings, and we use these in a model ß-sheet Hamiltonian to simulate amyloid vibrational spectra. We find that the variations in amyloid ß-sheet structures (e.g., dihedral angles, interstrand distances, and orientations) create significant differences in the average values for interstrand and nearest neighbor couplings, and that those variations encompass the variation in measured A⊥ frequencies. We also find that off-diagonal disorder about the average values explains the range of transition dipole strengths observed experimentally. Thus, we conclude that the lack of correlation between transition dipole-strength and frequency is caused by variations in amyloid ß-sheet structure. Taken together, these results indicate that the amide I frequency is very sensitive to amyloid ß-sheet structure, the ß-sheets of these 4 proteins are not identical, and the assumption that frequency of amyloids scales with ß-sheet size cannot be adopted without an accompanying measurement of transition dipole strengths.

Energy Transfer Between Coherently Delocalized States in Thin Films of the Explosive Pentaerythritol Tetranitrate (PETN) Revealed by Two-Dimensional Infrared Spectroscopy.

Pentaerythritol tetranitrate (PETN) is a common secondary explosive and has been used extensively to study shock initiation and energy propagation in energetic materials. We report 2D IR measurements of PETN thin films that resolve vibrational energy transfer and relaxation mechanisms. Ultrafast anisotropy measurements reveal a sub-500 fs reorientation of transition dipoles in thin films of vapor-deposited PETN that is absent in solution measurements, consistent with intermolecular energy transfer. The anisotropy is frequency dependent, suggesting spectrally heterogeneous vibrational relaxation. Cross peaks are observed in 2D IR spectra that resolve a specific energy transfer pathway with a 2 ps time scale. Transition dipole coupling calculations of the nitrate ester groups in the crystal lattice predict that the intermolecular couplings are as large or larger than the intramolecular couplings. The calculations match well with the experimental frequencies and the anisotropy, leading us to conclude that the observed cross peak is measuring energy transfer between two eigenstates that are extended over multiple PETN molecules. Measurements of the transition dipole strength indicate that these vibrational modes are coherently delocalized over at least 15-30 molecules. We discuss the implications of vibrational relaxation between coherently delocalized eigenstates for mechanisms relevant to explosives.

Watching Proteins Wiggle: Mapping Structures with Two-Dimensional Infrared Spectroscopy.

Proteins exhibit structural fluctuations over decades of time scales. From the picosecond side chain motions to aggregates that form over the course of minutes, characterizing protein structure over these vast lengths of time is important to understanding their function. In the past 15 years, two-dimensional infrared spectroscopy (2D IR) has been established as a versatile tool that can uniquely probe proteins structures on many time scales. In this review, we present some of the basic principles behind 2D IR and show how they have, and can, impact the field of protein biophysics. We highlight experiments in which 2D IR spectroscopy has provided structural and dynamical data that would be difficult to obtain with more standard structural biology techniques. We also highlight technological developments in 2D IR that continue to expand the scope of scientific problems that can be accessed in the biomedical sciences.

Spatially Resolved Two-Dimensional Infrared Spectroscopy via Wide-Field Microscopy.

We report the first wide-field microscope for measuring two-dimensional infrared (2D IR) spectroscopic images. We concurrently collect more than 16 000 2D IR spectra, made possible by a new focal plane array detector and mid-IR pulse shaping, to generate hyperspectral images with multiple frequency dimensions and diffraction-limited spatial resolution. Both frequency axes of the spectra are collected in the time domain by scanning two pairs of femtosecond pulses using a dual acousto-optic modulator pulse shaper. The technique is demonstrated by imaging a mixture of metal carbonyl absorbed polystyrene beads. The differences in image formation between FTIR and 2D IR microscopy are also explored by imaging a patterned USAF test target. We find that our 2D IR microscope has diffraction-limited spatial resolution and enhanced contrast compared to FTIR microscopy because of the nonlinear scaling of the 2D IR signal to the absorptivity coefficient for the vibrational modes. Images generated using off-diagonal peaks, created from vibrational anharmonicities, improve the molecular discrimination and eliminate noise. Two-dimensional wide-field IR microscopy provides information on vibrational lifetimes, molecular couplings, transition dipole orientations, and many other quantities that can be used for creating image contrast to help disentangle and interpret complex and heterogeneous samples. Such experiments made possible could include the study of amyloid proteins in tissues, protein folding in heterogeneous environments, and structural dynamics in devices employing mid-IR materials.

Experimental implementations of 2D IR spectroscopy through a horizontal pulse shaper design and a focal plane array detector.

Aided by advances in optical engineering, two-dimensional infrared spectroscopy (2D IR) has developed into a promising method for probing structural dynamics in biophysics and material science. We report two new advances for 2D IR spectrometers. First, we report a fully reflective and totally horizontal pulse shaper, which significantly simplifies alignment. Second, we demonstrate the applicability of mid-IR focal plane arrays (FPAs) as suitable detectors in 2D IR experiments. FPAs have more pixels than conventional linear arrays and can be used to multiplex optical detection. We simultaneously measure the spectra of a reference beam, which improves the signal-to-noise by a factor of 4; and two additional beams that are orthogonally polarized probe pulses for 2D IR anisotropy experiments.

Wide-field FTIR microscopy using mid-IR pulse shaping.

We have developed a new table-top technique for collecting wide-field Fourier transform infrared (FTIR) microscopic images by combining a femtosecond pulse shaper with a mid-IR focal plane array. The pulse shaper scans the delay between a pulse pair extremely rapidly for high signal-to-noise, while also enabling phase control of the individual pulses to under-sample the interferograms and subtract background. Infrared absorption images were collected for a mixture of W(CO)6 or Mn2(CO)10 absorbed polystyrene beads, demonstrating that this technique can spatially resolve chemically distinct species. The images are sub-diffraction limited, as measured with a USAF test target patterned on CaF2 and verified with scalar wave simulations. We also find that refractive, rather than reflective, objectives are preferable for imaging with coherent radiation. We discuss this method with respect to conventional FTIR microscopes.