TY - JOUR
T1 - Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry
AU - Calabrese, Antonio N.
AU - Jackson, Scott M.
AU - Jones, Lynsey N.
AU - Beckstein, Oliver
AU - Heinkel, Florian
AU - Gsponer, Joerg
AU - Sharples, David
AU - Sans, Marta
AU - Kokkinidou, Maria
AU - Pearson, Arwen R.
AU - Radford, Sheena E.
AU - Ashcroft, Alison E.
AU - Henderson, Peter J.F.
N1 - Funding Information:
This research was funded by the BBSRC (Grant No. BB/ K000659/1) to A.E.A., S.E.R., and P.J.F.H. including support for D.S. A.N.C. was supported by the Wellcome Trust Institutional Strategic Support Fund (ISSF; Grant No. 015615/Z/14/Z). A.E.A. and S.E.R. are also supported by the ERC under the EU’s Seventh Framework Programme (FP7.2007-2013/Grant No. 322408) and P.J.F.H. by an Emeritus Research Fellowship from the Leverhulme Trust (Grant No. EM-2014-045). S.M.J. and L.N.J. were supported by Research Studentships from the MRC and BBSRC, respectively. M.K. is supported by a Ph.D. studentship from the Joachim Herz Foundation. M.S. and A.R.P. are supported by the German Federal Excellence Cluster “The Hamburg Centre for Ultrafast Imaging”. J.G. and F.H. acknowledge funding from NSERC (Natural Sciences and Engineering Research Council of Canada). O.B. was supported by the National Institute of General Medical Sciences of the National Institutes of Health (Award No. R01GM118772). The Waters Synapt G2-Si and Xevo mass spectrometers were purchased with funding from the BBSRC (Grant No. BB/M012573/1), and fermenter and allied equipment for protein production were funded by the BBSRC (Grant No. MPSI BBS/B/14418), the Wellcome Trust (Grant No. JIF 062164/Z/00/Z), and the University of Leeds. The MD trajectories of Mhp1 were kindly provided by Sergei Noskov (University of Calgary). We thank members of the Henderson, Ashcroft, and Radford research groups for helpful discussions.
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/9/5
Y1 - 2017/9/5
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.analchem.7b01310
DO - 10.1021/acs.analchem.7b01310
M3 - Article
C2 - 28726379
AN - SCOPUS:85028936604
SN - 0003-2700
VL - 89
SP - 8844
EP - 8852
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 17
ER -