Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant

Antonello Calcutta, Christian M Jessen, Manja Annette Behrens, Cristiano L P Oliveira, Maria Lourdes Renart, José M González-Ros, Daniel Otzen, Jan Skov Pedersen, Anders Malmendal, Niels Christian Nielsen

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Abstract

Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl β-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE.
OriginalsprogEngelsk
TidsskriftB B A - Biomembranes
Vol/bind1818
Nummer9
Sider (fra-til)2290-301
Antal sider12
ISSN0005-2736
DOI
StatusUdgivet - 2012

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