Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry

Antonio N. Calabrese, Scott M. Jackson, Lynsey N. Jones, Oliver Beckstein, Florian Heinkel, Joerg Gsponer, David Sharples, Marta Sans, Maria Kokkinidou, Arwen R. Pearson, Sheena E. Radford, Alison E. Ashcroft, Peter J.F. Henderson

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion-substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein's conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.

Original languageEnglish (US)
Pages (from-to)8844-8852
Number of pages9
JournalAnalytical Chemistry
Volume89
Issue number17
DOIs
StatePublished - Sep 5 2017

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Dissection
Facings
Membrane Transport Proteins
Mass spectrometry
Cysteine
Conformations
Hydantoins
Substrates
Labeling
Sodium
Ions
Enzyme kinetics
Proteins
Sulfhydryl Compounds
Stoichiometry
Detergents
Membrane Proteins
Binding Sites
Ligands

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry. / Calabrese, Antonio N.; Jackson, Scott M.; Jones, Lynsey N.; Beckstein, Oliver; Heinkel, Florian; Gsponer, Joerg; Sharples, David; Sans, Marta; Kokkinidou, Maria; Pearson, Arwen R.; Radford, Sheena E.; Ashcroft, Alison E.; Henderson, Peter J.F.

In: Analytical Chemistry, Vol. 89, No. 17, 05.09.2017, p. 8844-8852.

Research output: Contribution to journalArticle

Calabrese, AN, Jackson, SM, Jones, LN, Beckstein, O, Heinkel, F, Gsponer, J, Sharples, D, Sans, M, Kokkinidou, M, Pearson, AR, Radford, SE, Ashcroft, AE & Henderson, PJF 2017, 'Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry', Analytical Chemistry, vol. 89, no. 17, pp. 8844-8852. https://doi.org/10.1021/acs.analchem.7b01310
Calabrese, Antonio N. ; Jackson, Scott M. ; Jones, Lynsey N. ; Beckstein, Oliver ; Heinkel, Florian ; Gsponer, Joerg ; Sharples, David ; Sans, Marta ; Kokkinidou, Maria ; Pearson, Arwen R. ; Radford, Sheena E. ; Ashcroft, Alison E. ; Henderson, Peter J.F. / Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry. In: Analytical Chemistry. 2017 ; Vol. 89, No. 17. pp. 8844-8852.
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AU - Heinkel, Florian

AU - Gsponer, Joerg

AU - Sharples, David

AU - Sans, Marta

AU - Kokkinidou, Maria

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