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

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

N-{1-[(3-Bromo­prop­yl)amino­carbon­yl]eth­yl}-2-(2-nitro­benzene­sulfonamido)propionamide

In the title compound, C15H21BrN4O6S, all three NH groups are involved in inter­molecular N—H⋯O inter­actions which, together with two inter­molecular C—H⋯O contacts, lead to a continuous anti­parallel β-sheet structure. There are no π–π inter­actions between mol­ecules, and two C—H⋯π inter­actions primarily govern the linkage between sheets

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

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

Cytochromeâ P450â Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs

Although membrane environment is known to boost drug metabolism by mammalian cytochromeâ P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptideâ based lipid nanodiscs to â trapâ the lipid boundaries of microsomal cytochromeâ P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The proteinâ induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.Redox catalysis in the lipid membrane: A novel application of peptide nanodiscs shows that cytochromeâ P450 2B4 is able to induce the formation of lipid raft domains in a biomimetic compound of the endoplasmic reticulum (ER). The proteinâ induced lipid rafts increase the thermal stability cytochromeâ P450 and dramatically alter the ligandâ binding kinetics of the hydrophilic ligand BHT.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142960/1/anie201713167.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142960/2/anie201713167_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142960/3/anie201713167-sup-0001-misc_information.pd

Synthesis, Characterization, and Nanodisc Formation of Non‐ionic Polymers**

Although lipid nanodiscs are increasingly used in the structural studies of membrane proteins, drug delivery and other applications, the interaction between the nanodisc belt and the protein to be reconstituted is a major limitation. To overcome this limitation and to further broaden the scope of nanodiscs, a family of non‐ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo‐oligosaccharides/inulin is reported. We show the stability of lipid nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic‐alignment properties. The reported results also demonstrate that the non‐ionic polymers extract membrane proteins with unprecedented efficiency.Hydrophobically functionalized fructo‐oligosaccharides/inulin polymers were synthesized and demonstrated to form lipid bilayer nanodiscs. These non‐ionic amphiphilic polymer‐based nanodiscs are stable against pH and divalent metal ions and they align magnetically in the presence of an external magnetic field. The reported results also demonstrate that the non‐ionic polymers extract membrane proteins with unprecedented efficiency.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/168431/1/ange202101950-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/168431/2/ange202101950_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/168431/3/ange202101950.pd