Chris T. Middleton

Two-dimensional infrared spectroscopy reveals the complex behaviour of an amyloid fibril inhibitor

While amyloid formation has been implicated in the pathology of over twenty human diseases, the rational design of amyloid inhibitors is hampered by a lack of structural information about amyloid-inhibitor complexes. We use isotope labeling and two-dimensional infrared spectroscopy to obtain a residue-specific structure for the complex of human amylin, the peptide responsible for islet amyloid formation in type 2 diabetes, with a known inhibitor, rat amylin. Based on its sequence, rat amylin should block formation of the C-terminal β-sheet, but at 8 hours after mixing rat amylin blocks the N-terminal β-sheet instead. At 24 hours after mixing, rat amylin blocks neither β-sheet and forms its own β-sheet most likely on the outside of the human fibrils. This is striking because rat amylin is natively disordered and not previously known to form amyloid β-sheets. The results show that even seemingly intuitive inhibitors may function by unforeseen and complex structural processes.

Development and Validation of Transferable Amide I Vibrational Frequency Maps for Peptides

Infrared (IR) spectroscopy of the amide I band has been widely utilized for the analysis of peptides and proteins. Theoretical modeling of IR spectra of proteins requires an accurate and efficient description of the amide I frequencies. In this paper, amide I frequency maps for protein backbone and side chain groups are developed from experimental spectra and vibrational lifetimes of N-methylacetamide and acetamide in different solvents. The frequency maps, along with established nearest-neighbor frequency shift and coupling schemes, are then applied to a variety of peptides in aqueous solution and reproduce experimental spectra well. The frequency maps are designed to be transferable to different environments; therefore, they can be used for heterogeneous systems, such as membrane proteins.

Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy.

We describe a methodology for studying protein kinetics using a rapid-scan technology for collecting 2D IR spectra. In conjunction with isotope labeling, 2D IR spectroscopy is able to probe the secondary structure and environment of individual residues in polypeptides and proteins. It is particularly useful for membrane and aggregate proteins. Our rapid-scan technology relies on a mid-IR pulse shaper that computer generates the pulse shapes, much like in an NMR spectrometer. With this device, data collection is faster, easier, and more accurate. We describe our 2D IR spectrometer, as well as protocols for (13)C(18)O isotope labeling, and then illustrate the technique with an application to the aggregation of the human islet amyloid polypeptide implicated in type 2 diabetes.

Base-stacking disorder and excited-state dynamics in single-stranded adenine homo-oligonucleotides.

Single-stranded adenine homo-oligonucleotides were investigated in aqueous solution by femtosecond transient absorption spectroscopy in order to study the effect of strand length on the nature and dynamics of excited states formed by UV absorption. Global fitting analysis of bleach recovery signals recorded at a probe wavelength of 250 nm and pH 7 reveals that the same lifetimes of 2.72 and 183 ps reproduce the pronounced biexponential decays observed in all (dA)n oligomers, containing between 2 and 18 residues. Although the lifetimes are invariant, the amplitudes of the short- and long-lived components depend sensitively on the number of residues. For example, the 183 ps component increases with strand length and is greater for DNA vs RNA single strands with the same number of adenines. Inhomogeneous kinetics arising from two classes of adenine bases in each oligomer best explains the observations. A subset of adenine residues produce short-lived excited states upon excitation, while absorption by the remaining adenines yields long-lived excited states that are responsible for the long-lived signal. By assuming that each short-lived excited state in the oligomer makes the same contribution to the transient absorption signal as an excited state of the adenine mononucleotide, the fraction of each type of base in the oligomer can be estimated along with the quantum yield of long-lived excited states. The fraction of oligonucleotides that yield long-lived excited states increases with oligomer length in precisely the same manner as the fraction of bases that are found in base stacks. Corroborating evidence that base stacking leads to distinct decay channels comes from experiments conducted at low pH on (dA)2. Coulombic repulsion between the two protonated bases at pH 2 results in open, unstacked conformations causing the long-lived component seen in (dA)2 at neutral pH to vanish completely. The fast component seen in oligomers with two or more bases is assigned to vibrational cooling following ultrafast internal conversion to the electronic ground state. This monomer-like decay channel is operative for the subset of adenine residues that are either poorly or not at all stacked with neighboring bases. This study shows that static base stacking disorder fully accounts for the length-dependent transient absorption signals. Although absorption likely creates delocalized excitons of unknown spatial extent, the results from this study suggest that long-lived excitations in single-stranded A tracts are already fully localized on no more than two bases no later than 1 ps after UV excitation.

Polarization shaping in the mid-IR and polarization-based balanced heterodyne detection with application to 2D IR spectroscopy.

We demonstrate amplitude, phase and polarization shaping of femtosecond mid-IR pulses using a germanium acousto-optical modulator by independently shaping the frequency-dependent amplitudes and phases of two orthogonally polarized pulses which are then collinearly overlapped using a wire-grid polarizer. We use a feedback loop to set and stabilize the relative phase of the orthogonal pulses. We have also used a wire-grid polarizer to implement polarization-based balanced heterodyne detection for improved signal-to-noise of 2D IR spectra collected in a pump-probe geometry. Applications include coherent control of molecular vibrations and improvements in multidimensional IR spectroscopy.

The Sulfated Triphenyl Methane Derivative Acid Fuchsin is a Potent Inhibitor of Amyloid Formation by Human Islet Amyloid Polypeptide and Protects Against the Toxic Effects of Amyloid Formation

Islet amyloid polypeptide (IAPP), also known as amylin, is responsible for amyloid formation in type 2 diabetes. The formation of islet amyloid is believed to contribute to the pathology of the disease by killing beta-cells, and it may also contribute to islet transplant failure. The design of inhibitors of amyloid formation is an active area of research, but comparatively little attention has been paid to inhibitors of IAPP in contrast to the large body of work on beta-amyloid, and most small-molecule inhibitors of IAPP amyloid are generally effective only when used at a significant molar excess. Here we show that the simple sulfonated triphenyl methane derivative acid fuchsin, 3-(1-(4-amino-3-methyl-5-sulfonatophenyl)-1-(4-amino-3-sulfonatophenyl) methylene) cyclohexa-1,4-dienesulfonic acid, is a potent inhibitor of in vitro amyloid formation by IAPP at substoichiometric levels and protects cultured rat INS-1 cells against the toxic effects of human IAPP. Fluorescence-detected thioflavin-T binding assays, light-scattering, circular dichroism, two-dimensional IR, and transmission electron microscopy measurements confirm that the compound prevents amyloid fibril formation. Ionic-strength-dependent studies show that the effects are mediated in part by electrostatic interactions. Experiments in which the compound is added at different time points during the lag phase after amyloid formation has commenced reveal that it arrests amyloid formation by trapping intermediate species. The compound is less effective against the beta-amyloid peptide, indicating specificity in its ability to inhibit amyloid formation by IAPP. The work reported here provides a new structural class of IAPP amyloid inhibitors and demonstrates the power of two-dimensional infrared spectroscopy for characterizing amyloid inhibitor interactions.

Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics

Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death. DOI: http://dx.doi.org/10.7554/eLife.12977.001

Mode selectivity with polarization shaping in the mid-IR.

We report that polarization-shaped mid-infrared (IR) pulses can be used to enhance the vibrational population of one mode over another in a coupled molecular system. A genetic algorithm and a new mid-IR polarization shaper were used to alter the relative vibrational excitation of the two carbonyl stretching modes in Mn(CO)(5)Br. One mode could be selectively enhanced over the other by 2-3 times. Control over the polarization leads to better optimization than phase-only control. Several possible mechanisms that indicate how polarization shaping leads to selective vibrational excitation are discussed using a formalism that separates polarization shaping effects on the signal strength from amplitude or phase shaping. The techniques introduced herein will have broad applications in quantum gating schemes, controlling ground state chemistry and enhancing the sensitivity of multidimensional IR and visible spectroscopies.

Fully absorptive 3D IR spectroscopy using a dual mid-infrared pulse shaper.

This paper presents the implementation of 3D IR spectroscopy by adding a second pump beam to a two-beam 2D IR spectrometer. An independent mid-IR pulse shaper is used for each pump beam, which can be programmed to collect its corresponding dimension in either the frequency or time-domains. Due to the phase matching geometry employed here, absorptive 3D IR spectra are automatically obtained, since all four of the rephasing and non-rephasing signals necessary to generate absorptive spectra are collected simultaneously. Phase cycling is used to isolate the fifth-order from the third-order signals. The method is demonstrated on tungsten hexacarbonyl (W(CO)6) and dicarbonylacetylacetonato rhodium (I), for which the eigenstates are extracted up to the third excited state. Pulse shaping affords a high degree of control over 3D IR experiments by making possible mixed time- and frequency-domain experiments, fast data acquisition and straightforward implementation.

Advances in Ultrafast 2-D Spectroscopy

This talk will cover recent developments in the use of femtosecond pulse shaping technology for collecting 2-D IR and 2-D Vis spectra and their applications to biophysical and energy related topics.