General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays.

This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable.

Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter.

Efflux pumps belonging to the ubiquitous resistance-nodulation-cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.