Self-assembled phospholipid bilayer Nanodiscs have become an important and versatile tool among model membrane systems to functionally reconstitute membrane proteins. Nanodiscs consist of lipid domains encased within an engineered derivative of apolipoprotein A-1 scaffold proteins, which can be tailored to yield homogeneous preparations of disks with different diameters, and with epitope tags for exploitation in various purification strategies. A critical aspect of the self-assembly of target membranes into Nanodiscs lies in the optimization of the lipid:protein ratio. Here we describe strategies for performing this optimization and provide examples for reconstituting bacteriorhodpsin as a trimer, rhodopsin, and functionally active P-glycoprotein. Together these demonstrate the versatility of Nanodisc technology for preparing monodisperse samples of membrane proteins of wide-ranging structure.

Phospholipid bilayer Nanodiscs are novel model membranes derived from high-density lipoprotein particles and have proven to be useful in studies of membrane proteins. Membrane protein enzymology has been hampered by the inherent insolubility of membrane proteins in aqueous environments and has necessitated the use of model membranes such as liposomes and detergent-stabilized micelles. Current model membranes display a polydisperse particle size distribution and can suffer from problems of inconsistency and instability. It is also unclear how well they mimic biological lipid bilayers. In contrast, Nanodiscs, the particle size of which is constrained by a coat of scaffold proteins, are relatively monodisperse, stable model membranes with a "nativelike" lipid bilayer. Nanodiscs have already been used to study a variety of membrane proteins, including cytochrome P450s, seven-transmembrane proteins, and bacterial chemoreceptors. These proteins are simultaneously monomerized, solubilized, and incorporated into the well-defined membrane environment provided by Nanodiscs. Nanodiscs may also provide useful insights into the thermodynamics and biophysics of biological membranes and binding of small molecules to membranes.

The cytochrome P-450 monooxygenase systems have received substantial attention due to their unique spectral and chemical properties.1,2 Of particular interest are the precise chemical mechanisms involved in the binding and activation of atmospheric dioxygen and the subsequent functionalization of an unactivated carbon substrate.3,4 We now report the first demonstration of a particular active site feature of cytochrome P-450 that is essential for efficient reduction and activation of molecular dioxygen, yet is independent of the substrate binding and spin-state equilibria processes. This has been accomplished by site-directed mutagenesis of threonine 252, a residue conserved among all known P-450 sequences, to an alanine (T252A),5 in the active site of the Pseudomonas cytochrome P-450cam.Although the individual cytochrome P-450 isozymes demonstrate marked differences in their substrate specificity,6 789predictions based on amino acid sequence analysis indicate that they share several conserved structural features.7-9 In particular, a long proximal -helix exhibits strong homology throughout a diverse cross section of the P-450s. The high-resolution X-ray crystal structure of cytochrome P-450cam10 *and molecular modeling *To whom reprint requests should be addressed.

Catalytic promiscuity is a widespread, but poorly understood, phenomenon among enzymes with particular relevance to the evolution of new functions, drug metabolism, and in vitro biocatalyst engineering. However, there is at present no way to quantitatively measure or compare this important parameter of enzyme function. Here we define a quantitative index of promiscuity (I) that can be calculated from the catalytic efficiencies of an enzyme toward a defined set of substrates. A weighted promiscuity index (J) that accounts for patterns of similarity and dissimilarity among the substrates in the set is also defined. Promiscuity indices were calculated for three different enzyme classes: eight serine and cysteine proteases, two glutathione S-transferase (GST) isoforms, and three cytochrome P450 (CYP) isoforms. The proteases ranged from completely specific (granzyme B, J ) 0.00) to highly promiscuous (cruzain, J ) 0.83). The four drug-metabolizing enzymes studied (GST A1-1 and the CYP isoforms) were highly promiscuous, with J values between 0.72 and 0.92; GST A4-4, involved in the clearance of lipid peroxidation products, is moderately promiscuous (J ) 0.37). Promiscuity indices also allowed for studies of correlation between substrate promiscuity and an enzyme's activity toward its most-favored substrate, for each of the three enzyme classes.

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHISNLC and aHISTPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.

The membrane-bound protein cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme. Most studies of ligand binding by CYP3A4 are currently carried out in solution, in the absence of a model membrane. Therefore, there is little information concerning the membrane effects on CYP3A4 ligand binding behavior. Phospholipid bilayer Nanodiscs are a novel model membrane system derived from high density lipoprotein particles, whose stability, monodispersity, and consistency are ensured by their self-assembly. We explore the energetics of four ligands (6-(p-toluidino)-2-naphthalenesulfonic acid (TNS), ␣-naphthoflavone (ANF), miconazole, and bromocriptine) binding to CYP3A4 incorporated into Nanodiscs. Ligand binding to Nanodiscs was monitored by a combination of environment-sensitive ligand fluorescence and ligand-induced shifts in the fluorescence of tryptophan residues present in the scaffold proteins of Nanodiscs; binding to the CYP3A4 active site was monitored by ligand-induced shifts in the heme Soret band absorbance. The dissociation constants for binding to the active site in CYP3A4-Nanodiscs were 4.0 M for TNS, 5.8 M for ANF, 0.45 M for miconazole, and 0.45 M for bromocriptine. These values are for CYP3A4 incorporated into a lipid bilayer and are therefore presumably more biologically relevant that those measured using CYP3A4 in solution. In some cases, affinity measurements using CYP3A4 in Nanodiscs differ significantly from solution values. We also studied the equilibrium between ligand binding to CYP3A4 and to the membrane. TNS showed no marked preference for either environment; ANF preferentially bound to the membrane, and miconazole and bromocriptine preferentially bound to the CYP3A4 active site. Cytochrome P450 3A4 (CYP3A4)2 is a major drug-metabolizing enzyme, and its ligand binding and catalysis are therefore of wide interest. CYP3A4 is an integral membrane protein, interacting with the membrane via an embedded N-terminal helix and other hydrophobic surface regions; however, most investigations of ligand binding are currently carried out in solution using recombinant protein with a partially truncated helical anchor, in the absence of any model membrane. Therefore, there is a critical lack of understanding of how the membrane environment affects ligand binding to CYP3A4. Membrane effects may be especially important when in vitro binding or kinetic data are extrapolated to predictions of in vivo pharmacokinetics.Model membranes can profoundly alter the ligand binding behavior of membrane proteins. For example, the choice of model membranes can shift the apparent affinity of a spider venom toxin for voltage-dependent K ϩ channels more than 4 orders of magnitude (1). Apart from the structural and dynamic effects of incorporation into a model membrane, competition for ligand binding between a model membrane and an incorporated protein can alter the apparent affinity and stoichiometry of ligand binding by the protein in equilibrium and kinetic experiments.Parry et al. (2) presented separate analytical ...

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Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1′-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the anti-epileptic drug carbamazepine (CBZ) with characteristic decreases in the 1′-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, MDZ and CBZ bound to CYP3A4 were probed using longitudinal T1 NMR relaxation and molecular docking with AutoDock 4.2. The distances calculated from the longitudinal T1 NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ molecules or an MDZ molecule and a CBZ molecule assume a stacked configuration within the CYP3A4 active site. In either case, the proton at position-4 of the MDZ molecule was closer to the heme than the protons of the 1′-CH3 group. In contrast, molecular docking of a single molecule of MDZ revealed that the molecule was preferentially oriented with the 1′-CH3 position closer to the heme than the 4-position. This study provides the first detailed molecular analysis of heterotropic and homotropic cooperativity of a human cytochrome P450 from an NMR-based model. Cooperativity of ligand binding through direct interaction between stacked molecules may represent a common motif for homotropic and heterotropic cooperativity.