Picturing the Membrane‐assisted Choreography of Cytochrome P450 with Lipid Nanodiscs

Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, and requires enzymatic partners as well as environmental factors that tune its complex dynamic activity. P450 and its reducing counterparts are membrane‐bound proteins which are believed to dynamically interact to form functional complexes. Increasing experimental evidence signifies the role (s) of protein‐lipid interactions in P450’s catalytic function and efficiency. The challenges posed by the membrane have severely limited high‐resolution understanding of the molecular interfaces of these interactions. Nevertheless, recent NMR studies have provided piercing insights into the dynamic structural interactions that enable the function of P450. In this review, we will discuss different biomimetic approaches relevant to unveil molecular interplays at the membrane, focusing on our recent work on lipid‐nanodiscs. We also highlight the need to expand the use of nanodiscs, and the power of a combination of cutting‐edge solution and solid‐state NMR techniques, to study the dynamic structures of P450 as well as other membrane‐proteins.Cytochrome P450 is organized as a catalytic metabolon. Experimental evidence discloses the role (s) of protein‐lipid interactions in P450’s catalytic function. In this review, the authors discuss biomimetic approaches relevant to unveil molecular interplays at the membrane interface, focusing on their recent work on lipid‐nanodiscs, and highlight the need to expand the use of nanodiscs to other membrane‐protein research areas.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146408/1/cphc201800444_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146408/2/cphc201800444.pd

Magnetic Alignment of Polymer Macro‐Nanodiscs Enables Residual‐Dipolar‐Coupling‐Based High‐Resolution Structural Studies by NMR Spectroscopy

Experimentally measured residual dipolar couplings (RDCs) are highly valuable for atomic‐resolution structural and dynamic studies of molecular systems ranging from small molecules to large proteins by solution NMR spectroscopy. Here we demonstrate the first use of magnetic‐alignment behavior of lyotropic liquid‐crystalline polymer macro‐nanodiscs (>20 nm in diameter) as a novel alignment medium for the measurement of RDCs using high‐resolution NMR. The easy preparation of macro‐nanodiscs, their high stability against pH changes and the presence of divalent metal ions, and their high homogeneity make them an efficient tool to investigate a wide range of molecular systems including natural products, proteins, and RNA.Korrekt ausgerichtet: Polymer‐Nanoscheiben werden als Ausrichtungsmedium für die Messung dipolarer Restkopplungen mit hochauflösender NMR‐Spektroskopie genutzt. Ihre einfache Herstellung, hohe Stabilität gegen pH‐Änderungen und die Anwesenheit zweiwertiger Metallionen sowie ihre hohe Homogenität machen sie zu einem effizienten Werkzeug für die Untersuchung vieler molekularer Systeme wie Naturstoffe, Proteine und RNA.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151827/1/ange201907655-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151827/2/ange201907655_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151827/3/ange201907655.pd

Semenâ derived amyloidogenic peptidesâ Key players of HIV infection

Misfolding and amyloid aggregation of intrinsically disordered proteins (IDPs) are implicated in a variety of diseases. Studies have shown that membrane plays important roles on the formation of intermediate structures of IDPs that can initiate (and/or speedâ up) amyloid aggregation to form fibers. The process of amyloid aggregation also disrupts membrane to cause cell death in amyloid diseases like Alzheimer’s disease and typeâ 2 diabetes. On the other hand, recent studies reported the membrane fusion properties of amyloid fibers. Remarkably, amyloidâ fibril formation by short peptide fragments of highly abundant prostatic acidicâ phosphatase (PAP) in human semen and are capable of boosting the rate of HIV infection up to 400,000â fold during sexual contact. Unlike the least toxic fully matured fibers of most amyloid proteins, the semenâ derived enhancer of virus infection (SEVI) amyloidâ fibrils of PAP peptide fragments are highly potent in rendering the maximum rate of HIV infection. This unusual property of amyloid fibers has witnessed increasing number of studies on the biophysical aspects of fiber formation and fiberâ membrane interactions. NMR studies have reported a highly disordered partial helical structure in a membrane environment for the intrinsically disordered PAP peptide that promotes the fusion of the viral membrane with that of host cells. The purpose of this review article is to unify and integrate biophysical and immunological research reported in the previous studies on SEVI. Specifically, amyloid aggregation, dramatic HIV infection enhancing properties, membrane fusion properties, high resolution NMR structure, and approaches to eliminate the enhancement of HIV infection of SEVI peptides are discussed.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144611/1/pro3395_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144611/2/pro3395.pd

Magnetic Alignment of Polymer Macro‐Nanodiscs Enables Residual‐Dipolar‐Coupling‐Based High‐Resolution Structural Studies by NMR Spectroscopy

Experimentally measured residual dipolar couplings (RDCs) are highly valuable for atomic‐resolution structural and dynamic studies of molecular systems ranging from small molecules to large proteins by solution NMR spectroscopy. Here we demonstrate the first use of magnetic‐alignment behavior of lyotropic liquid‐crystalline polymer macro‐nanodiscs (>20 nm in diameter) as a novel alignment medium for the measurement of RDCs using high‐resolution NMR. The easy preparation of macro‐nanodiscs, their high stability against pH changes and the presence of divalent metal ions, and their high homogeneity make them an efficient tool to investigate a wide range of molecular systems including natural products, proteins, and RNA.The right alignment: Polymer macro‐nanodiscs are used as a novel alignment medium for the measurement of residual dipolar couplings using high‐resolution NMR spectroscopy. Their easy preparation, high stability against pH changes and the presence of divalent metal ions, and high homogeneity make them an efficient tool for the investigation of a wide range of molecular systems including natural products, proteins, and RNA.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151864/1/anie201907655-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151864/2/anie201907655.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151864/3/anie201907655_am.pd

Bioinspired, Sizeâ Tunable Selfâ Assembly of Polymerâ Lipid Bilayer Nanodiscs

Polymerâ based nanodiscs are valuable tools in biomedical research that can offer a detergentâ free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 1.6â kDa SMAâ based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymerâ based nanodiscs are characterized by light scattering, NMR, FTâ IR, and TEM. A remarkable feature is that nanodiscs of different sizes, from nanometer to subâ micrometer diameter, can be produced by varying the lipidâ toâ polymer ratio. The smallâ size nanodiscs (up to ca. 30â nm diameter) can be used for solution NMR spectroscopy studies whereas the magneticâ alignment of macroâ nanodiscs (diameter of > ca. 40â nm) can be exploited for solidâ state NMR studies on membrane proteins.Discrete bilayers: Lipid bilayer nanodiscs of different size are formed by using modified styrene maleic acid coâ polymer. The smallâ size nanodiscs (up to ca. 30â nm diameter) can be used for solution NMR spectroscopy studies, whereas the magnetic alignment of largeâ size nanodiscs (or macroâ nanodiscs with a diameter of >40â nm) can be exploited for solidâ state NMR studies on membrane proteins.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138344/1/anie201705569.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138344/2/anie201705569_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138344/3/anie201705569-sup-0001-misc_information.pd

Formation of pH‐Resistant Monodispersed Polymer–Lipid Nanodiscs

Polymer lipid nanodiscs are an invaluable system for structural and functional studies of membrane proteins in their near‐native environment. Despite the recent advances in the development and usage of polymer lipid nanodisc systems, lack of control over size and poor tolerance to pH and divalent metal ions are major limitations for further applications. A facile modification of a low‐molecular‐weight styrene maleic acid copolymer is demonstrated to form monodispersed lipid bilayer nanodiscs that show ultra‐stability towards divalent metal ion concentration over a pH range of 2.5 to 10. The macro‐nanodiscs (>20 nm diameter) show magnetic alignment properties that can be exploited for high‐resolution structural studies of membrane proteins and amyloid proteins using solid‐state NMR techniques. The new polymer, SMA‐QA, nanodisc is a robust membrane mimetic tool that offers significant advantages over currently reported nanodisc systems.All under control: A poor tolerance to pH and divalent metal ions and a lack of control over size are major limitations of polymer nanodiscs. A modified styrene maleimide based polymer is demonstrated to form monodispersed nanodiscs with ultrahigh stability towards divalent metal ions over a pH range of 2.5 to 10.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141640/1/anie201712017_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141640/2/anie201712017-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141640/3/anie201712017.pd

Metalâ Chelated Polymer Nanodiscs for NMR Studies

Paramagnetic relaxation enhancement (PRE) is commonly used to speed up spin lattice relaxation time (T1) for rapid data acquisition in NMR structural studies. Consequently, there is significant interest in novel paramagnetic labels for enhanced NMR studies on biomolecules. Herein, we report the synthesis and characterization of a modified poly(styreneâ coâ maleic acid) polymer which forms nanodiscs while showing the ability to chelate metal ions. Cu2+â chelated nanodiscs are demonstrated to reduce the T1 of protons for both polymer and lipidâ nanodisc components. The chelated nanodiscs also decrease the proton T1 values for a waterâ soluble DNA Gâ quadruplex. These results suggest that polymer nanodiscs functionalized with paramagnetic tags can be used to speedâ up data acquisition from lipid bilayer samples and also to provide structural information from waterâ soluble biomolecules.Speeding up data acquisition: Design of a polymer nanodisc containing a DOTA chelator enables the utilization of the PRE effect in studies using lipid nanodiscs. This new technique can be applied to waterâ soluble biomolecules such as Gâ quadruplexes.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152508/1/anie201910118.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152508/2/anie201910118-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152508/3/anie201910118_am.pd

Effects of antidepressants on the conformation of phospholipid headgroups studied by solid-state NMR

The effect of tricyclic antidepressants (TCA) on phospholipid bilayer structure and dynamics was studied to provide insight into the mechanism of TCA-induced intracellular accumulation of lipids (known as lipidosis). Specifically we asked if the lipid–TCA interaction was TCA or lipid specific and if such physical interactions could contribute to lipidosis. These interactions were probed in multilamellar vesicles and mechanically oriented bilayers of mixed phosphatidylcholine–phosphatidylglycerol (PC–PG) phospholipids using 31 P and 14 N solid-state NMR techniques. Changes in bilayer architecture in the presence of TCAs were observed to be dependent on the TCA's effective charge and steric constraints. The results further show that desipramine and imipramine evoke distinguishable changes on the membrane surface, particularly on the headgroup order, conformation and dynamics of phospholipids. Desipramine increases the disorder of the choline site at the phosphatidylcholine headgroup while leaving the conformation and dynamics of the phosphate region largely unchanged. Incorporation of imipramine changes both lipid headgroup conformation and dynamics. Our results suggest that a correlation between TCA-induced changes in bilayer architecture and the ability of these compounds to induce lipidosis is, however, not straightforward as imipramine was shown to induce more dramatic changes in bilayer conformation and dynamics than desipramine. The use of 14 N as a probe was instrumental in arriving at the presented conclusions. Copyright © 2004 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34947/1/1327_ftp.pd