Sean M. Decatur

Differential effects of phe19 and phe20 on fibril formation by amyloidogenic peptide Aβ16–22 (Ac‐KLVFFAE‐NH2)

The sequence KLVFFAE (Aβ16–22) in Alzheimers β‐amyloid is thought to be a core β‐structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in β‐sheet. To elucidate the mechanism of the initial folding process, we undertook combined X‐ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized Aβ16–22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild‐type (WT) Aβ16–22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab‐like β‐crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30‐Å period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side‐chain dispositions for WT and 20CN, but not for 19CN. These X‐ray results and modeling imply that in the assembly of WT Aβ16–22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation. Proteins 2010.

Vibrational circular dichroism with isotopic substitution. Application to alanine rich peptides

Vibrational spectroscopy provides a means of monitoring conformational change that is selective for various types of conformations and is effective on a fast time scale to detect equilibrium populations of dynamic conformational states. Interpretation of IR and Raman spectra are primarily dependent on frequency shifts of characteristic local modes as perturbed by their conformational environment. These have been shown to be far from unique, yet, particularly in terms of detecting structural change, have proven useful [1]. Vibrational circular dichroism (VCD) has enhanced sensitivity to conformational type and, due to its interpretation being dependent on band shape, is less susceptible to error from frequency shift [2]. None-the-less all of these vibrational techniques depend on those residues having a similar conformation giving overlapping bands with similar spectral responses. This allows determination of the dominant secondary structural type and development of methods for quantifying the fractional contribution of each conformational type to the structure, particularly for globular proteins. Vibrational techniques do not have the ability to distinguish site-specific contributions, that is to determine which residues in a protein or peptide have a specific conformation.

Peptide Reptation as a Mechanism for Rearrangements within a β-Sheet Aggregate

Introduction Many neurodegenerative diseases are characterized by the accumulation of amyloid fibers in the brain, which can occur when a protein misfolds into an extended β-sheet conformation. The nucleation of these β-sheet aggregates is of particular interest, not only because this is the rate determining step towards fiber formation but also because early, soluble aggregate species may be the cytotoxic entities in many diseases. The soluble oligomeric intermediates are difficult to isolate and, due to their large size and dynamic nature, difficult to characterize using traditional biophysical techniques. For these reasons, many studies aimed at studying the aggregation process use small peptides, derived from full length proteins of interest, which also show amyloidogenic behavior. Among the peptides studied extensively include the NFGAIL sequence from the islet amyloid polypeptide, fragments of the prion protein which include the AGAAAAGA amyloidogenic region, and many different fragments of the Alzheimer’s Αβ peptide. In simulations of these peptides, ensembles of β-sheet oligomers are initially formed, including species which have a non-native hydrogen bonding registry or mix parallel and antiparallel organization of the strands. Rearrangements from rapidly formed disordered β-sheets to wellordered oligomers are likely an essential step for forming a template capable of nucleating growth into larger fibrils. Infrared (IR) spectroscopy is well suited for probing these systems. β-sheet aggregates give distinctive amide I bands in the IR spectrum, and the inclusion of specific isotope labels in the peptide (isotope-edited IR spectroscopy) gives residuelevel structural details on the peptide conformation, including the detailed registry of strands within the β-sheet [1,2]. In the case of residues 109-122 of the prion protein (peptide H1; Ac-MKHMAGAAAAGAVV-NH2), the initial β-sheet aggregates formed in solution lack a regular register between strands, and stable amyloid fibers only form after the β-strands of the peptide have adopted their equilibrium antiparallel β-sheet configuration with residue 117 in register across all strands [2]. Adoption of this register is required for the formation of stable, twisted fibers of aggregates [3]. In this paper, we present the kinetic details of the realignment of these β-strands from their fast-formed non-equilibrium structure with no regular register of the strands into the more ordered β-sheets capable of aggregating into stable fibers. This process is likely the nucleating step towards the formation of stable fibers.

Frequency Resolved and Heterodyned Femtosecond Infrared Echoes of Pep tides; Multiple Pulse Coherent Vibrational Analogues ofNMR

Two-dimensional (2D) infrared (IR) spectra of a dipeptide and a 13C substituted 20 unit peptide are obtained from heterodyned photon echoes (PE). Off-diagonal peaks reflect the coupling between amide groups. The two amide groups of acetylproline-NH2 show different vibrational dynamics.

Amide I Frequency as a Probe of Tertiary Structure: FTIR Studies of Helix Bundle Peptides

Infrared spectroscopy is extensively used to determine secondary structure content of peptides and proteins. The frequency, intensity, and line width of the amide I band of a peptide are commonly used to distinguish between alpha helix, beta sheet, and random coil [1]. However, tertiary structure also plays a role in determining IR spectral features. The amide I band is dependent on solvent environment; amide I frequencies of peptides which are exposed to water (and hydrogen bonded with solvent) can be distinguished from those in nonpolar environments [2]. However, there have been no systematic studies of the effects of tertiary structure changes on the amide I’ band of a polypeptide.

Stabilization of Helical Conformation in Model Peptides by 2,2,2-Trifluoroethanol: An FTIR Study

Despite the widespread use of TFE as a helix-stabilizing agent, the mechanism of its action is still widely debated. While mechanisms in which TFE binds to residues in the helical conformation and stabilizes the structure have been proposed [1], there is no evidence for direct interactions between TFE and hydrophobic side chains [2]. Alternatively, recent explanations of the TFE effect have focussed on the impact of TFE on the structure of water and its solvation of peptide groups. Three different mechanisms of helix stabilization by TFE involving solvation effect have been proposed. Based on studies of the effect of TFE on the conformation of alanine-rich helical peptides and intramolecular hydrogen bonding in salicylic acid, Luo and Baldwin proposed that desolvation of the backbone carbonyls in a helix strengthens intrahelical hydrogen bonding; the stronger hydrogen bonding increases the enthalpic stability of the helix versus random coil in TFE/water mixtures [3]. In studies of coiled-coil peptides, Kenstis and Sosnick have proposed that the increased solvent structure in TFE/water mixtures (as opposed to pure water) raises the energy of solvation of peptide backbone groups in the unfolded state; this indirectly enhances the stability of the helical state [4]. Recently, Cammers-Goodwin and co-workers have proposed that in pure water, there is a greater ordering of the solvent shell around the helix compared to the coil state, resulting in an unfavorable entropic change; by disrupting hydrogen bonding between the helix backbone and solvent, TFE reduces the solvent ordering which occurs upon helix formation, stabilizing the helix relative to the coil state [5].