A Simple Doublet Lens Design for Mid-Infrared Imaging.

Wide-field mid-infrared (MIR) hyperspectral imaging offers a promising approach for studying heterogeneous chemical systems due to its ability to independently characterize the molecular properties of different regions of a sample. However, applications of wide-field MIR microscopy are limited to spatial resolutions no better than ∼1 μm. While methods exist to overcome the classical diffraction limit of ∼λ/2, chromatic aberration from transmissive imaging reduces the achievable resolution.

β-Bracelets: Macrocyclic Cross-β Epitope Mimics Based on a Tau Conformational Strain.

The aggregation of misfolded tau into neurotoxic fibrils is linked to the progression of Alzheimer's disease (AD) and related tauopathies. Disease-associated conformations of filamentous tau are characterized by hydrophobic interactions between side chains on unique and distant β-strand modules within each protomer. Here, we report the design and diversity-oriented synthesis of β-arch peptide macrocycles composed of the aggregation-prone PHF6 hexapeptide of tau and the cross-β module specific to the AD tau fold.

Cross-α/β polymorphism of PSMα3 fibrils.

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α-forming peptide, phenol soluble modulin alpha 3 (PSMα3).

A theoretical analysis of coherent cross-peaks in polarization selective 2DIR for detection of cross-α fibrils.

The coupled amide-I vibrational modes in peptide systems such as fibrillar aggregates can often provide a wealth of structural information, although the associated spectra can be difficult to interpret. Using exciton scattering calculations, we characterized the polarization selective 2DIR peak patterns for cross-α peptide fibrils, a challenging system given the similarity between the monomeric and fibrillar structures, and interpret the results in light of recently collected 2D data on the cross-α peptide phenol soluble modulin α3. We find that stacking of α-helices into fibrils couples the bright modes across helical subunits, generating three new Bloch-like extended excitonic states that we designate A, E, and E.

Local Electric Fields in Aqueous Electrolytes.

Vibrational Stark shifts were explored in aqueous solutions of organic molecules with carbonyl- and nitrile-containing constituents. In many cases, the vibrational resonances from these moieties shifted toward lower frequency as salt was introduced into solution. This is in contrast to the blue-shift that would be expected based upon Onsager's reaction field theory.

Does liquid-liquid phase separation drive peptide folding?

Proline-arginine (PR) dipeptide repeats have been shown to undergo liquid-liquid phase separation and are an example of a growing number of intrinsically disordered proteins that can assemble into membraneless organelles. These structures have been posited as nucleation sites for pathogenic protein aggregation. As such, a better understanding of the effects that the increased local concentration and volumetric crowding within droplets have on peptide secondary structure is necessary.

Hydrogen Bond Exchange and Ca Binding of Aqueous -Methylacetamide Revealed by 2DIR Spectroscopy.

Cation effects on proteins have been a challenge to understand. Herein, we present two-dimensional infrared (2DIR) spectroscopic measurements, coupled with molecular dynamics and spectroscopic calculations, of -methylacetamide (NMA), a common model of the peptide backbone, in aqueous CaCl. The 2DIR spectra reveal that the dynamics of the amide carbonyl of NMA is dominated by exchange between two states of varying hydration, one possessing a structure similar to aqueous NMA and one that is dehydrated by one hydrogen bond.

Resolution Enhancement in Wide-Field IR Imaging and Time-Domain Spectroscopy Using Dielectric Microspheres.

Wide-field imaging through dielectric microspheres has emerged in recent years as a simple and effective approach for generating super-resolution images at visible wavelengths. We present, to our knowledge, the first demonstration that dielectric microspheres can be used in a wide-field infrared (IR) microscope to enhance the far field resolution. We have observed a substantial improvement in resolution and magnification when images are collected through polystyrene microspheres.

A Free Energy Barrier Caused by the Refolding of an Oligomeric Intermediate Controls the Lag Time of Amyloid Formation by hIAPP.

Transiently populated oligomers formed en route to amyloid fibrils may constitute the most toxic aggregates associated with many amyloid-associated diseases. Most nucleation theories used to describe amyloid aggregation predict low oligomer concentrations and do not take into account free energy costs that may be associated with structural rearrangements between the oligomer and fiber states. We have used isotope labeling and two-dimensional infrared spectroscopy to spectrally resolve an oligomeric intermediate during the aggregation of the human islet amyloid protein (hIAPP or amylin), the protein associated with type II diabetes.

Instantaneous ion configurations in the K+ ion channel selectivity filter revealed by 2D IR spectroscopy.

Potassium channels are responsible for the selective permeation of K ions across cell membranes. K ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose four K binding sites. Here, we report on the two-dimensional infrared (2D IR) spectra of a semisynthetic KcsA channel with site-specific heavy (CO) isotope labels in the selectivity filter.

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.

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.

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.

Two-dimensional sum-frequency generation (2D SFG) spectroscopy: summary of principles and its application to amyloid fiber monolayers.

By adding a mid-infrared pulse shaper to a sum-frequency generation (SFG) spectrometer, we have built a 2D SFG spectrometer capable of measuring spectra analogous to 2D IR spectra but with monolayer sensitivity and SFG selection rules. In this paper, we describe the experimental apparatus and provide an introduction to 2D SFG spectroscopy to help the reader interpret 2D SFG spectra. The main aim of this manuscript is to report 2D SFG spectra of the amyloid forming peptide FGAIL.

Mutational analysis of preamyloid intermediates: the role of his-tyr interactions in islet amyloid formation.

Islet amyloid polypeptide (IAPP or Amylin) is a 37-residue, C-terminally amidated pancreatic hormone, cosecreted with insulin that forms islet amyloid in type 2 diabetes. Islet amyloid formation is complex and characterizing preamyloid oligomers is an important topic because oligomeric intermediates are postulated to be the most toxic species produced during fibril formation. A range of competing models for early oligomers have been proposed.

Two-dimensional sum-frequency generation reveals structure and dynamics of a surface-bound peptide.

Surface-bound polypeptides and proteins are increasingly used to functionalize inorganic interfaces such as electrodes, but their structural characterization is exceedingly difficult with standard technologies. In this paper, we report the first two-dimensional sum-frequency generation (2D SFG) spectra of a peptide monolayer, which are collected by adding a mid-IR pulse shaper to a standard femtosecond SFG spectrometer. On a gold surface, standard FTIR spectroscopy is inconclusive about the peptide structure because of solvation-induced frequency shifts, but the 2D line shapes, anharmonic shifts, and lifetimes obtained from 2D SFG reveal that the peptide is largely α-helical and upright.

Native state conformational heterogeneity of HP35 revealed by time-resolved FRET.

The villin headpiece subdomain (HP35) has become one of the most widely used model systems in protein folding studies, due to its small size and ultrafast folding kinetics. Here, we use HP35 as a test bed to show that the fluorescence decay kinetics of an unnatural amino acid, p-cyanophenylalanine (Phe(CN)), which are modulated by a nearby quencher (e.g.

Spectroscopic studies of protein folding: linear and nonlinear methods.

Although protein folding is a simple outcome of the underlying thermodynamics, arriving at a quantitative and predictive understanding of how proteins fold nevertheless poses huge challenges. Therefore, both advanced experimental and computational methods are continuously being developed and refined to probe and reveal the atomistic details of protein folding dynamics and mechanisms. Herein, we provide a concise review of recent developments in spectroscopic studies of protein folding, with a focus on new triggering and probing methods.

Direct assessment of the α-helix nucleation time.

The nucleation event in α-helix formation is a fundamental process in protein folding. However, determining how quickly it takes place based on measurements of the relaxation dynamics of helical peptides is difficult because such relaxations invariably contain contributions from various structural transitions such as from helical to nonhelical states and helical to partial-helical conformations. Herein, we measure the temperature-jump (T-jump) relaxation kinetics of three model peptides that fold into a single-turn α-helix, using time-resolved infrared spectroscopy, aiming to provide a direct assessment of the helix nucleation rate.

The Two Dimensional Vibrational Echo of a Nitrile Probe of the Villin HP35 Protein.

2D IR spectroscopy was used to probe the hydrophobic core structure of the 35-residue Villin headpiece subdomain, HP35, by monitoring the C≡N vibrational stretching band of a cyano substituted phenylalanine (Phe). The presence of two humps in the vibrational frequency distribution in the folded equilibrium state is revealed. They represent two states that exchange more slowly than ca.

Photophysics of a Fluorescent Non-natural Amino Acid: p-Cyanophenylalanine.

The non-natural amino acid p-cyanophenylalanine (Phe(CN)) has recently emerged as a useful fluorescent probe of proteins; however, its photophysical properties have not been systematically examined. Herein, we measure the fluorescence quantum yield and the fluorescence lifetime of Phe(CN) in a series of solvents. It is found that the fluorescence lifetime of Phe(CN) shows a linear dependence on the Kamlet-Taft parameter α of the protic solvents used, indicating that the solute-solvent hydrogen bonding interactions mediate the non-radiative decay rate.

The electronic role of DNA-functionalized carbon nanotubes: efficacy for in situ polymerization of conducting polymer nanocomposites.

We have found that the polymerization process was 4,500 times faster when a self-doped polyaniline nanocomposite was fabricated using in situ polymerization in the presence of single-stranded DNA-dispersed and -functionalized single-walled carbon nanotubes (ssDNA-SWNTs). More importantly, the quality of the composite was significantly improved: fewer short oligomers were produced, and the self-doped polyaniline backbone had a longer conjugation length and existed in the more stable and conductive emeraldine state. The functionality of the boronic acid group in the composite and the highly improved electronic performance may lead to broad applications of the composite in flexible electronic devices.