Refining Disordered Peptide Ensembles with Computational Amide I Spectroscopy: Application to Elastin-Like Peptides

Abstract

The characterization of intrinsically disordered protein (IDP) ensembles is complicated both by inherent heterogeneity and by the fact that many common experimental techniques function poorly when applied to IDPs. For this reason, the development of alternative structural tools for probing IDP ensembles has attracted considerable attention. Here we describe our recent work in developing experimental and computational tools for characterizing IDP ensembles using Amide I (backbone carbonyl stretch) vibrational spectroscopy. In this approach, the infrared (IR) absorption frequencies of isotope-labeled amide bonds probe their local electrostatic environments and structures. Empirical frequency maps allow us to use this spectroscopic data as a direct experimental test of atomistic structural models. We apply these methods to a family of short elastin-like peptides (ELPs), fragments of the elastin protein based around the Pro-Gly turn motif characteristic of the elastomeric segments of the full protein. Using a maximum entropy analysis of experimental spectra on the basis of predicted spectra from molecular dynamics (MD) ensembles, we find that peptides with Ala or Val sidechains preceding the Pro-Gly turn unit exhibit a stronger tendency toward extended structures than do Gly-Pro-Gly motifs, suggesting an important role for steric interactions in tuning the molecular properties of elastin.

Refinement of Peptide Conformational Ensembles by 2D IR Spectroscopy: Application to Ala‒Ala‒Ala

“…Our objective in this study is to test an ensemble refinement scheme that can be used to account for complex spectral features such as those found in FTIR and 2D IR spectra. In our recent study of ELPs, the ME refinement is proved effective for describing the extension in a type-I turn across multiple FFs (5). However, the constraints of mean frequency and variance of a spectrally isolated peak is not easily extended to more complex features such as multiple overlapping resonances and asymmetric spectral line shapes.…”

“…Among these factors, we found that the most significant factor is the error due to the frequency map, as partly seen in the systematic bias present in different water models above. Taking 2s (54 cm À1 ) of the spectroscopic map error as the estimated confidence interval (5,53), the highest uncertainty of determining the ensemble populations against the full set of spectra is found to be 18% in AMBERfb15 ensembles (Table S4). These high uncertainties indicate that the frequency error is the most influential source of degrading quality of the ensemble refinement.…”

“…Intrinsically disordered proteins (IDPs) exhibit a variety of thermally accessible conformers reflecting basins on a complex energy landscape that are also characterized by dynamics such as conformational fluctuations and activated kinetics of interconversion between free-energy basins (1)(2)(3). As a result, structural characterization of IDPs or proteins with intrinsic disordered regions requires an ensemble description, which creates numerous experimental challenges (4,5). In particular, ensemble-structure determination is naturally an ill-posed problem in which the degrees of freedom in relevant conformational states far exceeds the limited number of measurements and information content of experiments (6).…”

“…Enhanced structural information can be achieved by using various polarizations of ultrafast infrared pulses (17) and introducing site-specific isotope labeling such as 13 C or 13 C 18 O on the amide carbonyl, which provides an additional frequency shift to isolate a specific C¼O bond from a congested spectrum, allowing the extraction of local structural details (18,19). The structural sensitivity of amide I vibrational and site-specific isotope labeling has assisted in addressing the conformational distributions of peptides and proteins (5,(20)(21)(22), helix-coil transition dynamics (23)(24)(25), and the ion-permeation mechanism of ion channels (26,27).…”

“…Also, the recent efforts of force field (FF) developments have improved the ability to study disordered proteins (28), but the question of how much the uncertainty from FFs and water models affects the ensemble predictions of IDPs and proteins with intrinsic disordered regions quantitatively still remains (29)(30)(31)(32)(33). A practical approach using ensemble refinement, which reweights existing ensemble populations from simulations against experimental data, has proven successful for facilitating the inference of protein conformational ensembles consistent with experiments (6), including developing different frameworks such as the maximum entropy (ME) principle (34)(35)(36)(37)(38), Bayesian statistics (39)(40)(41), and biological applications against experimental data such as NMR (6,42), small angle x-ray scattering (43,44), and IR spectroscopy (5,45). A recent detailed review of ensemble structure determination can be found in (4).…”