Strategies for Extracting Structural Information from 2D IR Spectroscopy of Amyloid: Application to Islet Amyloid Polypeptide

Abstract

The 37-residue human islet amyloid polypeptide (hIAPP or amylin) self-assembles into fibers, the assembly of which has been associated with the disease mechanism of type II diabetes. Infrared spectroscopy in conjunction with isotope labeling is proving to be a powerful tool for studying the aggregation process of hIAPP and other amyloid forming proteins with residue specific structure and kinetic information, but the relationship between the spectroscopic observables and the structure is not fully established. We report a detailed analysis of the linear and 2D IR spectra of hIAPP fibers isotope labeled at seven different residue positions. The features of the 2D IR spectra, including the frequencies, linewidths, intensities, and polarization dependence of the diagonal and cross-peaks, rely heavily on the position of the isotope labeled residue. In order to understand how these measured parameters depend on fiber secondary and tertiary structure, we have simulated 1D and 2D IR spectra utilizing idealized structural models in addition to a recently published solid-state NMR based model of the amyloid fibril. The analysis provides a more rigorous foundation for interpreting the infrared spectra of amyloids. In addition, we demonstrate that 2D IR spectra can be employed to distinguish between residues in β-sheets versus those in turn regions, and that transitional residues between secondary structures can be identified by the suppression of their cross-peaks in 2D IR spectra. This latter approach is not limited to amyloid fibrils and will be generally useful in identifying regions of secondary structure in proteins using 2D IR spectroscopy and isotope labeling.

Vibrational Coupling between Helices Influences the Amide I Infrared Absorption of Proteins: Application to Bacteriorhodopsin and Rhodopsin

“…This fact has recently been recognized for the case of β -sheet structures. [20][21][22] The effect of coupling between secondary structure elements such as helices could be so large that the resulting band position falls outside the typical band assignment ranges. This could for instance be the case for BR, where a large number of helices in the biological trimer unit couple and shifts the band position to higher wavenumber.…”

“…12,13 The interactions within a helix have been well characterized during the past decades. [13][14][15][16][17][18][19] Several recent studies have instead focused on the IR spectral features of transition dipole coupling (TDC) between secondary structure elements, such as those between β -sheets [20][21][22] or α-helices in a helix-dimer. 23,24 This coupling is important to consider as it influences the band positions associated with the secondary structure absorption and therefore the interpretation of the IR spectra.…”

Quantification of transition dipole strengths using 1D and 2D spectroscopy for the identification of molecular structures via exciton delocalization: Application to α-helices

“…Thus, transition dipoles are good indicators of peptide secondary structure, for example, since many individual amide I bonds are coupled together in betasheets and α-helices, creating strong transition dipoles. 22,[25][26][27] Of course, one can also detect coupling by the frequency separation, but frequency shifts are not always easily measurable. In the example above, the coupling caused each peak to shift by only 1.5 cm −1 , which may be difficult to experimentally detect considering that the linewidths of most condensed phase vibrational modes are >10 cm −1 .…”

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

“…For an IR probe that is able to interact with water via H bonding, measurement of its FFCF can provide, sometimes in a site-specific manner, detailed information about the hydration dynamics of the protein molecule of interest. For example, this approach has been used to identify the existence of mobile water molecules inside Aβ40 amyloid fibrils (31,32) and to interrogate the water-assisted drug-binding mechanism of HIV-1 reverse transcriptase (33), among many other applications (34)(35)(36)(37)(38)(39). In the current study, we capitalize on the established sensitivity of the nitrile stretching vibration (C≡N) to local hydration and electrostatic environment (40) and use the unnatural amino acid p-cyano-phenyalanine (Phe CN ) as a local IR reporter.…”