Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution

Abstract

Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form ␣-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the ␣-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis.

Ab Initio Modeling of Amide I Coupling in Antiparallel β-Sheets and the Effect of 13C Isotopic Labeling on Infrared Spectra

“…1,6,9,28 Deuteration of solvent additionally removes the coupling and spectral overlap of water vibrations with the amide I mode by shifting the water bending absorption maximum from ∼1640 cm -1 (for H 2 O) to ∼1195 cm -1 (for D 2 O). 28,63,64 Empirical Modeling.…”

“…Studies of proteinprotein interactions have also been proposed by use of uniform labeling for one member of an interacting system so that the changes in each protein/peptide could be monitored separately. [25][26][27] We have previously studied infrared absorption (IR) and vibrational circular dichroism (VCD) of a series of R-helical peptides to determine localized sites of unfolding and the degree of coupling between various sites in the sequence 9,14 and have modeled these with quantum mechanically determined spectral parameters computed for smaller oligomers with constrained (φ, ψ) angles and transferred onto larger peptides with the same conformation. [28][29][30] The degree of coupling between amide groups is the essential physical interaction that makes amide vibrational spectra capable of determining conformation for a polymeric system.…”

“…In the past decade a number of studies have appeared where isotopic substitution was used to obtain better structural insight with IR spectra. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] Isotopic labeling of the amide group has an important impact on vibrational spectra, because it causes a shift of absorption frequencies and thus allows separation of transitions associated with the labeled residue from those of the rest of the molecule. Thus the properties of this transition reflect the local stereochemistry of the labeled group and its environment.…”

Molecular dynamics with restrictions derived from optical spectra

“…The vibrational circular dichroism (VCD) 5 and the Raman optical activity (ROA) 6 gave unique information about structure of many organic compounds, proteins, or nucleic acids. 7,8 Other important ways to increase effective resolution of the optical spectra include site-specific labeling with stable isotopes 9 and twodimensional multiphoton spectra sampling the chromophore coupling. 10 Simulations by modern quantum-chemical methods represent powerful tools for the spectra interpretations.…”

Cross-Strand Coupling and Site-Specific Unfolding Thermodynamics of a Trpzip β-Hairpin Peptide Using 13C Isotopic Labeling and IR Spectroscopy

“…The resulting thermal denaturation curves are often characterized by very broad transitions. [43][44][45][46][47] Increase in the number of interstrand hydrophobic interactions has been shown to stabilize -hairpin structures and result in somewhat more sigmoidal denaturation profiles in trpzips; 1,30,48 however, there can be a tendency for such peptide sequences to aggregate. 24 Due to their small size, trpzips have also been a subject of many theoretical studies.…”