Structure Characterization of an Intrinsically Disordered Peptide Using In-Droplet Hydrogen Deuterium Exchange Mass Spectrometry and Molecular Dynamics Simulations

Behaving allosterically in nature and possessing versatile structural conformational types, intrinsically disordered proteins (IDPs) as well as intrinsically disordered regions (proteins) exhibit multi-functional properties in living organisms. However, their dynamic nature composed of conformational fluctuations over very brief timescales has made characterizing solution states challenging. In this research, for the first time, a method to facilitate the conformational mapping of challenging peptide systems has been derived from in-droplet hydrogen/deuterium exchange and mass spectrometry (HDX-MS) data combined with molecular dynamics (MD) simulations. Field-enabled capillary vibrating sharp-edge sharp spray ionization (cVSSI) devices are coupled to MS to measure HDX reactions for structurally varied peptide systems including: polyalanine (PA), bradykinin (BK), model peptide (KDD), polyserine (PS), and the first 17 amino acid residues of the Exon 1 region of Huntingtin protein (Nt17) in the presence of internal standard (e.g., lysine and arginine). Initially, we determined that propensity factor of different heteroatoms hydrogen which characterized the hydrogen type in peptide of interests. Here, we have reported 86% deuterium incorporation of lysine in electro-osmotically favored mixing of analyte and D2O droplet in-droplet HDX measurements. From the estimated backbone amide deuterium incorporation data, MD simulations were harnessed to interpret and predict HDX behavior for peptides in a systematic fashion. Initially, a sequence-based intrinsic rate approach, which takes into account steric and electronic effects on backbone amide sites, showed a poor ability (correlation coefficient of 0.27) to predict backbone deuterium incorporation. A second approach which included the consideration of secondary and tertiary structure protection as well as the ability to form intra- and inter-molecular hydrogen bonds provided improved reaction prediction capability (correlation coefficient values of 0.79 and 0.90, respectively). Further modifying the model by including scaled relative intrinsic rates significantly improves reaction predictability (correlation coefficient values of 0.97 and 0.99, respectively). The newly developed model provides a crucial link between experimental and theoretical realms allowing interpretation of HDX-MS data. Remarkably, the novel approach suggests a strong protective role for structural rigidity along with secondary structure protection accounting for decreased deuterium incorporation especially for BK. Furthermore, application of the model to the Nt17 system suggests that, although the peptide may be considered as intrinsically disordered in nature, the high frequency formation of very transient secondary structure across the peptide sequence (overall higher inter-residue contact) render it relatively inflexible to HDX. That said, such structural fluctuation may also provide clues about the ability of the amphipathic Nt17 peptide to rapidly stabilize helical structure upon interaction with binding partners (lipid and/or peptide) and thus participate in aggregation phenomena associated with Huntington’s Disease (HD). The results lay the groundwork for further improvements of the model and its potential application in the study of IDP and IDR structural properties for many important proteins.