Abstract
Obtaining (dynamic) structure related information on proteins is key for understanding their function. Methods as single-molecule Förster Resonance Energy Transfer (smFRET) and Electron Paramagnetic Resonance (EPR) that measure distances between labeled residues to obtain dynamic information rely on selection of suitable residue pairs for chemical modification. Selection of pairs of amino acids, that show sufficient distance changes upon activity of the protein, can be a tedious process. Here we present an in silico approach that makes use of two or more structures (or structure models) to filter suitable residue pairs for FRET or EPR from all possible pairs within the protein. We apply the method for the study of the conformational dynamics of the substrate-binding domain of the osmoregulatory ATP-Binding Cassette transporter OpuA. This method speeds up the process of designing mutants, and because of its systematic nature, the chances of missing promising candidates are reduced.
Highlights
Obtaining structure related information on proteins is key for understanding their function
Selected labelling positions are already available for this protein, which have been used in previous single-molecule Förster Resonance Energy Transfer (smFRET) studies[10]
The V360 and N423 are present in the lobes of OpuAC that interact with the transmembrane domain (TMD) of OpuA19
Summary
Obtaining (dynamic) structure related information on proteins is key for understanding their function. Methods as single-molecule Förster Resonance Energy Transfer (smFRET) and Electron Paramagnetic Resonance (EPR) that measure distances between labeled residues to obtain dynamic information rely on selection of suitable residue pairs for chemical modification. Double electron–electron resonance (DEER) or Pulsed Electron–electron double resonance (PELDOR) can be used to probe distance changes upon changing c onditions[5] and single-molecule Förster Resonance Energy Transfer (smFRET) can be used to obtain single molecule dynamics of proteins and other macromolecular a ssemblies[6,7,8,9,10] The latter two techniques make use of two labels that are introduced for instance by attaching them to cysteine residues via maleimide[11] or methanethiosulfonate c hemistry[12] or introducing them as non-natural amino a cids[13]. We tested the method by designing pairs for labelling in the substrate-binding domain (OpuAC) of the ABC transporter OpuA and performed single-molecule FRET measurements and functionality assays of the full-length protein complex
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