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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. A structural rearrangement includes the F23G24A25I26L27 region of hIAPP, which starts from a random coil structure, evolves into ordered β-sheet oligomers containing at least 5 strands, and then partially disorders in the fibril structure. The supercritical concentration is measured to be between 150 and 250 μM, which is the thermodynamic parameter that sets the free energy of the oligomers. A 3-state kinetic model fits the experimental data, but only if it includes a concentration independent free energy barrier >3 kcal/mol that represents the free energy cost of refolding the oligomeric intermediate into the structure of the amyloid fibril; i.e., "oligomer activation" is required. The barrier creates a transition state in the free energy landscape that slows fibril formation and creates a stable population of oligomers during the lag phase, even at concentrations below the supercritical concentration. Largely missing in current kinetic models is a link between structure and kinetics. Our experiments and modeling provide evidence that protein structural rearrangements during aggregation impact the populations and kinetics of toxic oligomeric species.

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. Here we describe the design and implementation of a simple MIR achromatic lens combination that we believe will aid in the development of resolution-enhanced wide-field MIR hyperspectral optical and chemical absorption imaging. We also examine the use of this doublet lens to image through polystyrene microspheres, an emerging and simple means for enhancing spatial resolution. [ABSTRACT FROM AUTHOR]

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. The role of the amidated C-terminus in amyloid formation by IAPP and in stabilizing oligomers is not known. Studies with unamidated IAPP have provided evidence for formation of an antiparallel dimer at pH 5.5, stabilized by stacking of His-18 and Tyr-37, but it is not known if this interaction is formed in the physiological form of the peptide. Analysis of a set of variants with a free and with an amidated C-terminus shows that disrupting the putative His-Tyr interaction accelerates amyloid formation, indicating that it is not essential. Amidation to generate the physiologically relevant form of IAPP accelerates amyloid formation, demonstrating that the advantages conferred by C-terminal amidation outweigh increased amyloidogenicity. The analysis of this variant argues that IAPP is not under strong evolutionary pressure to reduce amyloidogenicity. Analysis of an H18Q mutant of IAPP shows that the charge state of the N-terminus is an important factor controlling the rate of amyloid formation, even though the N-terminal region of IAPP is believed to be flexible in the amyloid fibers.

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⊥. Coherent superpositions of these states in broadband 2DIR simulations lead to characteristic signals that are sensitive to fibril length and match the experimental 2DIR spectra. [ABSTRACT FROM AUTHOR]

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 α-helical structure of these peptides is stabilized by a covalent cross-linker formed between the side chains of two residues at the iand i 4 positions. If we assume that this cross-linker mimics the structural constraint arising from a strong side chain–side chain interaction (e.g., a salt bridge) in proteins, these peptides would represent good models for studying the nucleation process of an α-helix in a protein environment. Indeed, we find that the T-jump induced relaxation rate of these peptides is approximately (0.6 μs)−1at room temperature, which is slower than that of commonly studied alanine-based helical peptides but faster than that of a naturally occurring α-helix whose folded state is stabilized by a series of side chain–side chain interactions. Taken together, our results put an upper limit of about 1 μs for the helix nucleation time at 20 °C and suggest that the subsequent propagation steps occur with a time constant of about 240 ns. [ABSTRACT FROM AUTHOR]

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 (13C18O) isotope labels in the selectivity filter. The ultrafast time resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by molecular dynamics simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites. [ABSTRACT FROM AUTHOR]

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. Herein we use Fourier transform infrared (FTIR) and two-dimensional infrared (2DIR) spectroscopy to show that formation of droplets by PR 20 accompanies changes in the amide-I spectra consistent with folding into poly-proline helical structures., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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 N -methylacetamide (NMA), a common model of the peptide backbone, in aqueous CaCl 2 . 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. In addition, we demonstrate that at high (>5 M) CaCl 2 concentrations, direct binding of Ca 2+ to the carbonyl of NMA occurs, stabilizing an iminium-type resonance structure of NMA, with a characteristic C═N + stretch frequency at 1680 cm -1 . This species is only marginally populated and is only detectable in 2DIR spectra due to its larger transition strength.