Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry. Calabrese, A. N., Jackson, S. M., Jones, L. N., Beckstein, O., Heinkel, F., Gsponer, J., Sharples, D., Sans, M., Kokkinidou, M., Pearson, A. R., Radford, S. E., Ashcroft, A. E., & Henderson, P. J. F. Analytical Chemistry, 89(17):8844–8852, September, 2017.
Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry [link]Paper  doi  abstract   bibtex   
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.
@article{calabrese_topological_2017,
	title = {Topological {Dissection} of the {Membrane} {Transport} {Protein} {Mhp1} {Derived} from {Cysteine} {Accessibility} and {Mass} {Spectrometry}},
	volume = {89},
	issn = {0003-2700},
	url = {http://dx.doi.org/10.1021/acs.analchem.7b01310},
	doi = {10.1021/acs.analchem.7b01310},
	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.},
	number = {17},
	urldate = {2018-01-17},
	journal = {Analytical Chemistry},
	author = {Calabrese, Antonio N. and Jackson, Scott M. and Jones, Lynsey N. and Beckstein, Oliver and Heinkel, Florian and Gsponer, Joerg and Sharples, David and Sans, Marta and Kokkinidou, Maria and Pearson, Arwen R. and Radford, Sheena E. and Ashcroft, Alison E. and Henderson, Peter J. F.},
	month = sep,
	year = {2017},
	pages = {8844--8852},
}
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