title: Ion Mobility Spectrometry-Mass Spectrometry of Intrinsically Unfolded Proteins: Trying to Put Order into Disorder.
abstract: Intrinsically disordered proteins do not adopt well-defined native structures and therefore present an intriguing challenge in terms of structural elucidation as they are relatively inaccessible to traditional approaches such as NMR and X-ray crystallography. Many members of this important group of proteins have a distinct biological function and frequently undergo a conformational change on binding to their physiological targets which can in turn modulate their function. Furthermore, many intrinsically unstructured proteins are associated with a wide range of major diseases including cancer and amyloid-related disorders. Here, electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to probe the conformational characteristics of two intrinsically disordered proteins:apo-cytochrome c andapo-osteocalcin. Both proteins are structured in theirholo-states when bound to their respective substrates, but disordered in theirapo-states. Here, the conformational properties of theholo- and theapo-protein forms for both species have been analysed and their mass spectral data and ion mobility spectrometry-derived collision cross-sectional areas, indicative of their physical size, compared to study the relationship between substrate binding and tertiary structure. In both cases, the intrinsically unstructuredapo-states populated multiple conformations with larger cross-sectional areas than theirholo-analogues, suggesting that intrinsic disorder in proteins does not preclude the formation of preferred conformations. Additionally, analysis of truncated analogues of osteocalcin has located the region of the protein responsible for the conformational changes detected upon metal cation binding. Together, the data illustrate the scope and utility of ESI-IMS-MS for studying the characteristics and properties of intrinsically disordered proteins whose analysis by other techniques is limited.
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Mathematics and Physical Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: T W
authors.initials: TW
authors.lastname: Knapman
authors.forename: N M
authors.initials: NM
authors.lastname: Valette
authors.forename: S L
authors.initials: SL
authors.lastname: Warriner
authors.forename: A E
authors.initials: AE
authors.lastname: Ashcroft
keywords: Collision cross-sectional areas; cytochrome c; electrospray ionisation; intrinsically disordered proteins; ion mobility spectrometry; mass spectrometry; osteocalcin.