Search

Amyloid aggregates are highly ordered fibrillar assemblies of polypeptides involved in a number of neurodegenerative diseases. Very little is known on the pathways of self-assembly of peptides into the final amyloid fibrils, which is due in part to the difficulty of triggering the aggregation process in a controlled manner. Here we present the design and validation of a cross-linked hexapeptide that reversibly aggregates and dissociates under ultraviolet light irradiation control. First molecular dynamics simulations were carried out to identify, among hundreds of possible sequences, those with the highest propensity to form ordered (β-sheet) oligomers in the trans state of the azobenzene cross-linker, and at the same time with the highest solubility in the cis state. In the simulations, the peptides were observed to spontaneously form ordered oligomers with cross-β contacts when the cross-linker was in the trans state, whereas in the cis state they self-assemble into amorphous aggregates. For the most promising sequence emerging from the simulations (Ac-Cys-His-Gly-Gln-Cys-Lys-NH(2) cross-linked at the two cysteine residues), the photoisomerization of the azobenzene group was shown to induce reversible aggregation by time-resolved light scattering and fluorescence measurements. The amyloid-like fibrillar topology was confirmed by electron microscopy. Potential applications of minimally designed peptides with photoswitchable amyloidogenic propensity are briefly discussed.

We present two-dimensional infrared (2D IR) spectra of the OD stretch vibration of isotope diluted water (HOD/H(2)O) from ambient conditions (293 K) down to the metastable supercooled regime (260 K). We observe that spectral diffusion slows down from 700 fs to 2.6 ps as we lower the temperature. A comparison between measurements performed at the magic angle with those at parallel polarization shows that the 2D IR line shape is affected by the frequency-dependent anisotropy decay in the case of parallel polarization, altering the extracted correlation decay. A fit within the framework of an Arrhenius law reveals an activation energy of E(a) = 6.2 ± 0.2 kcal/mol and a pre-exponential factor of 1/A = 0.02 ± 0.01 fs. Alternatively, a power law fit results in an exponent γ = 2.2 and a singularity temperature T(s) = 221 K. We tentatively conclude that the power law provides the better physical picture to describe the dynamics of liquid water around the freezing point.

A series of photoswitchable, alpha-helical peptides were studied using two-dimensional infrared spectroscopy (2D-IR). Single-isotope labeling with (13)C(18)O at various positions in the sequence was employed to spectrally isolate particular backbone positions. We show that a single (13)C(18)O label can give rise to two bands along the diagonal of the 2D-IR spectrum, one of which is from an amide group that is hydrogen-bonded internally, or to a solvent molecule, and the other from a non-hydrogen-bonded amide group. The photoswitch enabled examination of both the folded and unfolded state of the helix. For most sites, unfolding of the peptide caused a shift of intensity from the hydrogen-bonded peak to the non-hydrogen-bonded peak. The relative intensity of the two diagonal peaks gives an indication of the fraction of molecules hydrogen-bonded at a certain location along the sequence. As this fraction varies quite substantially along the helix, we conclude that the helix is not uniformly folded. Furthermore, the shift in hydrogen bonding is much smaller than the change of helicity measured by CD spectroscopy, indicating that non-native hydrogen-bonded or mis-folded loops are formed in the unfolded ensemble.

Vibrational energy transport in a short 3(10)-helical peptide is studied by time-resolved femtosecond infrared spectroscopy. The C-D vibrations of decadeuterated leucine incorporated in the helical chain are excited, and the subsequent flow of vibrational energy through the helix is monitored by employing C horizontal lineO probes at various distances from the heat source as local thermometers. The C-D modes are not resonant to the C horizontal lineO modes, neither directly nor through any Fermi resonance, thereby suppressing resonant energy transfer directly along the C horizontal lineO oscillators of the peptide backbone. In contrast to our previous work (J. Phys. Chem. B 2008, 112, 9091), we no longer find any substantial difference in the vibrational energy transport efficiency after high- or low-energy excitation. That is, the heat diffusion constant of (2.0 +/- 0.5) A(2) ps(-1) is the same as that after depositing vibrational energy through the ultrafast internal conversion of a covalently bound chromophore.

We demonstrate the extension of the principle of difference Fourier transform infrared (FTIR) spectroscopy to difference 2D-IR spectroscopy. To this end, we measure difference 2D-IR spectra of the protein bacteriorhodopsin in its early J- and K-intermediates. By comparing with the static 2D-IR spectrum of the protonated Schiff base of all-trans retinal, we demonstrate that the 2D-IR spectrum of the all-trans retinal chromophore in bacteriorhodopsin can be measured with the background from the remainder of the protein completely suppressed. We discuss several models to interpret the detailed line shape of the difference 2D-IR spectrum.

Two cyclic disulfide-bridged tetrapeptides [cyclo(Boc-Cys-Pro-Aib-Cys-OMe) (1) and cyclo(Boc-Cys-Pro-Phe-Cys-OMe) (2)] have been monitored by time-resolved mid-IR spectroscopy in the C=O vibrational range. A conformational change is induced by cleavage of the intramolecular disulfide bridge upon UV excitation (lambda(exc) = 260 nm), giving rise to a pair of cysteinyl radicals (thiyl radicals), which diffuse apart allowing the peptide to change conformation before they undergo quenching. The amide I band reports on the dynamics of the peptide backbone, which evolves on a 100 ps time scale and then stays constant up to 10 micros at low enough concentrations ( approximately 100 mM). To probe specifically the lifetime of the free cysteinyl radicals, time-resolved UV laser flash photolysis has been applied. The concentration of the cysteinyl radical decays nonexponentially, but about 50% are still present after 1 ms. The photocleavable disulfide bridge hence may serve as an intrinsic, naturally occurring phototrigger to study peptide dynamics that opens a wide time-window from a few picoseconds to many hundreds of microseconds.

The technique of transient two-dimensional infrared (T2D-IR) spectroscopy is introduced, which extends the advantage of 2D-IR spectroscopy to the investigation of a transient species with picosecond time resolution. The conformational change of a small cyclic peptide is studied in the amide-I spectral range, which is induced by means of a photoswitch integrated into the peptide backbone. Substantial changes are found in the transient 2D-IR spectra at times when the transient 1D spectra show only a minor time dependence, illustrating the information gain accessible from 2D-IR spectroscopy. In contrast to 1D spectroscopy, 2D-IR can distinguish between homogeneous and inhomogeneous broadening. The homogeneous contribution to the total width of the amide-I band changes during the course of the conformational transition, a result that is interpreted in terms of the manner in which the peptide samples its conformational space.

Using polarization sensitive two-dimensional (2D) vibrational spectroscopy on the amide I mode, the central backbone structure of trialanine in aqueous solution is investigated. We exploit the polarization sensitivity of the 2D pump−probe signal to reveal the cross-peak structure hidden under the strong diagonal peaks. The dihedral angles φ and ψ characterizing the peptide backbone structure are derived directly from the cross-peak intensity and anisotropy, demonstrating the potential of 2D spectroscopy as a tool for peptide structure elucidation.

Femtosecond infrared (IR) pump probe and dynamic hole burning experiments were used to examine the ultrafast response of the modes in the 1600−1700 cm-1 region (the so-called amide I modes) of N-methylacetamide (NMA) and three small globular peptides, apamin, scyllatoxin, and bovine pancreatic trypsin inhibitor (BPTI). A value of 16 cm-1 was found for the anharmonicity of the amide I vibration. Vibrational relaxation of the amide I modes of all investigated peptides occurs in ca. 1.2 ps. An even faster value of 450 fs is obtained for NMA, a model for the peptide unit. The vibrational relaxation is dominated by intramolecular energy redistribution (IVR) and reflects an intrinsic property of the peptide group in any environment. Dynamic hole burning experiments with a narrow band pump pulse which selectively excites only a subset of the amide I eigenstates reveal that energy migration between different amide I states is slow compared with vibrational relaxation. Two-dimensional pump−probe (2D-IR) spectra th...