Joshua S. Ostrander

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.

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.

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.

Spectroscopic Signature for Stable β-Amyloid Fibrils versus β-Sheet-Rich Oligomers

We use two-dimensional IR (2D IR) spectroscopy to explore fibril formation for the two predominant isoforms of the β-amyloid (Aβ1-40 and Aβ1-42) protein associated with Alzheimers disease. Two-dimensional IR spectra resolve a transition at 1610 cm-1 in Aβ fibrils that does not appear in other Aβ aggregates, even those with predominantly β-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 Aβ 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 Aβ crystal structures. It was not previously possible to distinguish stable Aβ fibrils from the less stable β-sheet-rich oligomers with infrared light. This 2D IR signature will be useful for Alzheimers research on Aβ aggregation, fibril formation, and toxicity.

Not All β-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 β-sheet amide I mode for the aggregated proteins amyloid-β1-40, calcitonin, α-synuclein, and glucagon. According to standard vibrational coupling models for proteins, the frequencies of canonical β-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 β-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 β-sheet Hamiltonian to simulate amyloid vibrational spectra. We find that the variations in amyloid β-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 β-sheet structure. Taken together, these results indicate that the amide I frequency is very sensitive to amyloid β-sheet structure, the β-sheets of these 4 proteins are not identical, and the assumption that frequency of amyloids scales with β-sheet size cannot be adopted without an accompanying measurement of transition dipole strengths.

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.