Membrane-bound cytochrome P4503A4 (CYP3A4) is the major source of enzymatic drug metabolism. Although several structural models of CYP3A4 in various ligand complexes are available, none includes a lipid bilayer. Details of the effects of the membrane on protein dynamics and solvation, and access channels for ligands, remain uncertain. H/D exchange mass spectrometry (H/DXMS) with ligand free CYP3A4 containing a deletion of residues 3–12, compared to that of the full length wild type, in lipid nanodiscs afforded 91% sequence coverage. Deuterium exchange was fast in the F- and G-helices, HI loop, and C-terminal loop. In contrast, there is very low exchange in the F′-and G′-helices. The results are consistent with the overall membrane orientation of CYP3A4 suggested by published MD simulations and spectroscopic results, and the solvent accessibility of the F/G loop suggests that it is not deeply membrane-embedded. Addition of ketoconazole results in only modest, but global, changes in solvent accessibility. Interestingly, with ketoconazole bound some peptides become less solvent accessible or dynamic, including the F- and G-helices, but several peptides demonstrate modestly increased accessibility. Differential scanning calorimetry (DSC) of CYP3A4-nanodiscs suggests membrane-induced stabilization compared to that of aggregated CYP3A4 in buffer, and this stabilization is enhanced upon addition of the ligand ketoconazole. This ligand-induced stabilization is accompanied by a very large increase in ΔH for CYP3A4 denaturation in nanodiscs, possibly due to increased CYP3A4–membrane interactions. Together, the results suggest a distinct orientation of CYP3A4 on the lipid membrane, and they highlight likely solvent access channels, which are consistent with several MD simulations.

Lipid nanodiscs provide a native‐like lipid environment for membrane proteins, and they have become a valuable platform for the study of membrane biophysics. A range of biophysical and biochemical analyses are enabled when membrane proteins are captured in lipid nanodiscs. Two parameters that can be controlled when capturing membrane proteins in lipid nanodiscs are the radius, and hence the surface area of the lipid surface, and the composition of the lipid bilayer. Despite their emergence as a versatile tool, most studies with lipid nanodiscs in the literature have focused on nanodiscs of a single radius with a single lipid. In light of the complexity of biological membranes, it is likely that nanodiscs with multiple membrane components would be more sophisticated models for membrane research. It is possible to prepare nanodiscs with more complex lipid mixtures to probe the effects of lipid composition on several aspects of membrane biochemistry. Detailed protocols are described here for the preparation of nanodiscs with mixtures of phospholipids, incorporation of cholesterol, and incorporation of a spectroscopic lipid probe. These protocols provide starting points for the construction of nanodiscs with more physiological membrane compositions or with useful biophysical probes. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Assembly of mixed lipid nanodiscs Basic Protocol 2: Assembly of nanodiscs with cholesterol Basic Protocol 3: Incorporation of laurdan into nanodiscs for membrane fluidity measurements

Methods for the initial steps of surface plasmon resonance analysis of membrane proteins incorporated in lipid nanodiscs are described. Several types of BiacoreTM sensor chips are available and require distinct strategies to immobilize proteonanodiscs on the chip surface. The procedures for immobilization on three of these chips (NTA, antibody coupled CM5, and L1) are described and results are demonstrated for a model system with cytochrome P4503A4 (CYP3A4) in nanodiscs binding to a polyclonal anti-CYP3A4 antibody. Advantages and disadvantages of each chip type are considered.

Especially in developing countries, the impact of vaccines can be limited by logistical obstacles associated with multiple dose regimens, pathogen variants, and challenges imposed by requirements for maintaining vaccines at low temperatures during shipping and storage. Thus, there is a need for vaccines that can be flexibly modified to address evolving pathogen landscapes, are stable outside of narrow "cold-chain" temperatures and require administration of only single doses. Here we demonstrate in proof-of-concept studies a vaccine platform that addresses these impediments to more widespread use of vaccines. The platform relies on bacteriophage-derived phage-like-particles (PLPs) that utilize a "plug-and-play" antigen delivery system that allows for fast, easy alteration of antigens on the surface of the PLPs. Thermostability of PLP-based vaccines can be achieved by embedding the PLPs within glassy particles produced by spray drying, and nanoscopic aluminum oxide layers applied using atomic layer deposition (ALD) can serve to control release of antigen in vivo, yielding vaccine formulations that elicit strong immune responses after administration of single doses. Bacteriophage λ was stabilized by spray drying to form powders that were incubated at 37 °C for up to a year without loss of infectious activity. PLPs derived from bacteriophage λ were expressed and purified from E. coli cultures, and an in vitro conjugation strategy was used to decorate specific PLP surface sites with T4-lysozyme, a model vaccine antigen. The resulting T4-lysozyme:PLP complexes (Lys-PLPs) were embedded in glassy dry powders formed by spray drying and coated with nanometer-thick layers of alumina deposited by ALD in a fluidized bed reactor. Alumina-coated Lys-PLP vaccines were stable for a least a month at 50 °C, and single doses of the alumina-coated vaccines elicited immune responses that were indistinguishable from responses generated by conventional two-dose, prime-and-boost dosing regimens of alum-adjuvanted Lys-PLP vaccines.

Biological therapeutics represent an increasing and critical component of newly approved drugs; however, the inability to deliver biologics intracellularly in a controlled manner remains a major limitation. We have developed a semi-synthetic, tunable phage-like particle (PLP) platform derived from bacteriophage λ. The shell surface can be decorated with small-molecule, biological and synthetic moieties, alone or in combination and in defined ratios. Here, we demonstrate that the platform can be used to deliver biological macromolecules intracellularly and in a controlled manner. Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that has been widely recognized as an ideal target for the treatment of a variety of cancers. Recently, UbV.7.2, a novel biologic derived from the ubiquitin scaffold, was developed for inhibition of USP7, but issues remain in achieving efficient and controlled intracellular delivery of the biologic. We have shown that decoration of PLPs with trastuzumab (Trz), a HER2-targeted therapeutic used in the treatment of various cancers, results in specific targeting and uptake of Trz-PLPs into HER2-overexpressing breast cancer cells. By simultaneously decorating PLPs with Trz and UbV.7.2, we now show that these particles are also internalized by HER2-positive cells, thus providing a means for intracellular delivery of the biologic in a controlled fashion. Internalized particles retain USP7 inhibition activity of UbV.7.2 and alter the metabolic and proteomic landscapes of these cells. This study demonstrates that the λ "designer nanoparticles" represent a powerful system for the intracellular delivery of biologics in a defined dose.