Crystallogenesis of Membrane Proteins Mediated by Polymer-Bounded Lipid Nanodiscs  Jana Broecker, Bryan T. Eger, Oliver P. Ernst  Structure  Volume 25, Issue 2, Pages 384-392 (February 2017) DOI: 10.1016/j.str.2016.12.004 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 384-392DOI: (10.1016/j.str.2016.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Detergent-Based versus Polymer-Mediated in Meso Crystallogenesis Comparison of membrane-protein crystallization in the LCP following (left/blue) traditional detergent-based protein solubilization and purification or (right/yellow) polymer-based protein solubilization and purification retaining a bilayer environment. As proof of principle, a seven-transmembrane α-helical membrane protein is produced in E. coli and either solubilized and purified with detergent (blue; step 1) or solubilized and purified with SMA copolymers (yellow; step 1). Solubilized proteins are purified chromatographically (step 2) and transferred into the LCP (step 3) for growth of crystals (step 4). Protein structures are solved by X-ray crystallography (step 5). Lipid molecules are shown in green. Structure 2017 25, 384-392DOI: (10.1016/j.str.2016.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Biophysical Characterization of HwBR in OG Micelles or SMA Nanodiscs Results of solubilization optimization with SMA are given in Figures S1A and S1B. (A) SDS PAGEs reflecting the purity of both preparations after Ni2+-based IMAC and SEC with the HwBR band (∼29.3 kDa) and some impurities (∗,∗∗) indicated (M, marker; MP, main peak; SP, side peak). For a preparation with higher purity see Figure S1C. (B) SEC profiles for HwBR in OG micelles versus SMA nanodiscs (A, absorbance; V, volume). The latter are larger and less homogeneous than HwBR–OG complexes. (C) UV-visible absorbance spectra of purified HwBR. SMA copolymers contribute significantly to the A280nm signal. Absorbance at ∼420 nm is likely due to co-purified soluble hemeproteins (λ, wavelength). (D) Intensity-weighted size distributions obtained from dynamic light scattering (I, intensity; d, hydrodynamic diameter). Structure 2017 25, 384-392DOI: (10.1016/j.str.2016.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Diffraction Quality of Crystals Generated by the Polymer-Mediated Approach Crystals obtained from protein purified in SMA nanodiscs and grown in the LCP diffract to a resolution better than 2.0 Å. (A) Typical X-ray diffraction pattern as detected using a PILATUS detector at the Advanced Photon Source at Argonne National Laboratory (Lemont, IL, USA). Note that a pattern with one well-defined and one diffuse ring is characteristic of crystals grown in the LCP. Three typical intense, round spots are shown at the right (frame colors match those in the complete pattern). (B) Four examples of reflection spots (arrows) with a resolution better than 2.0 Å that can still be seen by the naked eye. Any standard indexing software is even more sensitive than visual inspection and will detect more spots at resolutions better than those indicated. Crystal images are shown in Figure S2. Structure 2017 25, 384-392DOI: (10.1016/j.str.2016.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Structural Alignment of HwBR Monomers Structural alignment of HwBR monomers obtained from LCP-grown crystals that originated either from protein solubilized in DDM micelles and purified in OG micelles (green; all-trans-retinal shown in blue) or solubilized and purified in SMA nanodiscs (magenta; all-trans-retinal shown in orange) with a Cα RMSD of 0.22 Å for the trimer in the asymmetric unit. (A–D) HwBR pumps protons from the intracellular side (intra) to the extracellular side (extra) (A). Parts of the proton translocation path with functional residues in stick representation are indicated as follows: (B) retinal-binding pocket with all-trans-retinal bound to Lys224 by a Schiff base linkage and with proton re-uptake residue Asp93, (C) proton-releasing complex (note that the Glu202 side chain shows some flexibility in orientation between different protomers), and (D) proton outward cap region with hydrogen bonds shown as gray dashes. (E) The 2Fo – Fc electron density maps of retinal and the surrounding residues are contoured at 1.0σ and shown in blue (detergent–LCP) and orange (SMA–LCP). Comparison and structural alignment of structures obtained from the detergent-based or polymer-mediated approaches are given in Figure S3. Crystallization and crystal information as well as data collection and refinement statistics are given in Tables S1 and S2. Structure 2017 25, 384-392DOI: (10.1016/j.str.2016.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions