Direct cell extraction of membrane proteins for structure–function analysis

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Membrane proteins are the largest group of therapeutic targets in a variety of disease areas and yet, they remain particularly difficult to investigate. We have developed a novel one-step approach for the incorporation of membrane proteins directly from cells into lipid Salipro nanoparticles. Here, with the pannexin1 channel as a case study, we demonstrate the applicability of this method for structure–function analysis using SPR and cryo-EM.

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  1. Review coordinated via ASAPbio’s crowd preprint review

    This review reflects comments and contributions by Ruchika Bajaj, Sree Rama Chaitanya Sridhara and Sara El Zahed. Review synthesized by Ruchika Bajaj.

    This study has developed a novel one-step methodology for the incorporation of membrane proteins from cells to lipid Salipro nanoparticles for structure-function studies using surface plasmon resonance (SPR) and single-particle cryoelectron microscopy (cryo-EM), which is a profound technology in the field of membrane protein structural biology. We raise some points that may strengthen the manuscript below:

    • Main section, 4th paragraph “resuspended in digitoxin-containing buffer”- Does the sentence mean that membrane proteins were solubilized by detergent before reconstitution into salipro particles? Are salipro and digitoxin added at the same step? If this is the case, it is unclear how one can distinguish between the step wise solubilization and reconstitution or direct reconstitution into salipro particles. Further discussion on the mechanism of reconstitution would be helpful. In the same paragraph, the fragment “to increase membrane fluidity and render lipids” raises the question of whether the concentration of digitonin was optimized to balance the increase in membrane fluidity but not rendering the solubilization of membrane proteins.
    • Main section, 4th paragraph, “the formation of saponin-containing mPANX1-GFP particles was assessed by analytical size exclusion chromatography using fluorescence detector” - It is assumed that fluorescence is detected from GFP. As the construct expressed is PANX1-GFP, GFP fluorescence signal will be received from reconstituted as well as not reconstituted PANX1. Is saponin specific signal being used as a signal for measuring the reconstitution of PANX1-GFP? In the same paragraph, “PreScission protease for on-column cleavage” is mentioned. Is GFP still intact in the expressed PANX-1 or is it cleaved? A diagram of these procedures showing the various steps will be helpful for readers.
    • Main section, 4th paragraph “SDS-PAGE revealed the formation of pure and homogeneous Salipro-mPANX1 nanoparticles”- However, extra bands are present above the major band in Figure 1E, can some comment be provided on this point. Possible explanations for the additional bands could be post translational modifications or degradation of mPANX1.
    • Methodology section, “membrane protein reconstitution screening using fluorescence-detection size exclusion chromatography (FSEC)” - The amount of salipro is given in ug. A comment on the ratio of protein to salipro particles would be important to decide the concentration of salipro with respect to the mass of the cell pellet.
    • Figure 1G: The molecular weight of Salipro-mPANX1 particles is mentioned to be approximately 466kD. mPANX1 weighs about 48kD and heptamer will be 336kDa. A discussion on comparison of experimental and actual molecular weight would be interesting.
    • hPANX1 was expressed in sf9 insect cells. A description regarding trials of expression of this construct in expi293 cells would be informative.
    • Supplemental Figure 1B: The gel is overloaded and shows multiple bands for hPANX1, recommend selecting an alternative image for hPANX.
    • Paragraph 6A phrase, “challenged with bezoylbenzoyl-ATP(bzATP), spironolactone and cabenoxolone” - Please explain the meaning of ‘challenged’ here.
    • Supplementary Figure 2: Paragraph 6 mentions “binding constant could not be determined”. Please provide an explanation for this. Is it about the saturation phase not being approachable because of the feasibility of the binding experiment at higher concentration of cabenoxolone?
    • The last summary sentence in Paragraph 6 is not clear, recommend rephrasing it.
    • Figure 2A shows that Salipro particles have His tag. This suggests that an additional step of affinity purification with His tag could have been used to distinguish or separate reconstituted and un-reconstituted PANX1.
    • Supplementary figure 4: Please explain whether the datasets for samples in the presence and absence of fluorinated lipids were combined together.
    • Paragraph 8, “intracellular helices were not well resolved” - Please comment on a possible explanation. Does the Salipro scaffold contribute to the resolution? Please mention any future possibilities regarding improving the resolution by modifying the salipro scaffold or alternative scaffold. In the same paragraph, rmsd is mentioned at promoter level, please comment on how this value changes at heptamer level and why is it important to report the rmdd value to appreciate the direct reconstitution methodology.
    • Last paragraph 10, “future membrane protein research” - Please comment on the utility of this methodology on prokaryotic membrane proteins, bacterial outer or inner membrane proteins or eukaryotic membrane proteins. Some more examples of reconstitution with the same method will support the applicability of this methodology on diverse kinds of membrane proteins. A discussion section comparing this methodology to other methods would also be useful for readers.