Guy Patchornik

Structural Organization and Energy Transduction Mechanism of Na+,K+-ATPase Studied with Transition Metal-Catalyzed Oxidative Cleavage

This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na+,K+-ATPase to our understanding of structure–function relations. In the presence of ascorbate/H2O2, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe2+ bound at the cytoplasmic surface or by complexes of ATP–Fe2+, which directs the Fe2+ to the normal ATP–Mg2+ site. Fe2+- and ATP–Fe2+-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg2+ ions between E1P and E2P, which may be crucial in facilitating hydrolysis of E2P. The pattern of domain interactions in E1 and E2 conformations, and role of Mg2+ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu2+ ions, bound at the extracellular surfaces, or a hydrophobic Cu2+-diphenyl phenanthroline (DPP) complex, which directs the Cu2+ to the membrane–water interface. Cu2+- or Cu2+-DPP-catalyzed cleavages are providing information on α/β subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.

Purification of a Membrane Protein with Conjugated Engineered Micelles

A novel method for purifying membrane proteins is presented. The approach makes use of engineered micelles composed of a nonionic detergent, β-octylglucoside, and a hydrophobic metal chelator, bathophenanthroline. Via the chelators, the micelles are specifically conjugated, i.e., tethered, in the presence of Fe(2+) ions, thereby forming micellar aggregates which provide the environment for separation of lipid-soluble membrane proteins from water-soluble proteins. The micellar aggregates (here imaged by cryo-transmission electron microscopy) successfully purify the light driven proton pump, bacteriorhodopsin (bR), from E. coli lysate. Purification takes place within 15 min and can be performed both at room temperature and at 4 °C. More than 94% of the water-soluble macromolecules in the lysate are excluded, with recovery yields of the membrane protein ranging between 74% and 85%. Since this approach does not require precipitants, high concentrations of detergent to induce micellar aggregates, high temperature, or changes in pH, it is suggested that it may be applied to the purification of a wide variety of membrane proteins.

Tethered non-ionic micelles: a matrix for enhanced solubilization of lipophilic compounds

A specific mechanism for tethering micelles composed of non-ionic detergents is presented. The mechanism does not require any precipitant, high ionic strength or temperature alterations. Rather, it relies on complexes between hydrophobic chelators embedded within the micelle and appropriate metal cations in the aqueous phase, serving as mediators. The approach was applied to: (i) four non-ionic detergents (tetraethylene glycol monooctyl ether (C8E4), n-dodecyl-β-D-maltoside (DDM), octyl β-D-1-thioglucopyranoside (OTG), and n-octyl-β-D-glucopyranoside (OG)), (ii) two hydrophobic chelators (bathophenanthroline and N-(1,10-phenanthrolin-5-yl)decanamide, Phen-C10) and (iii) five transition metals (Fe2+, Ni2+, Zn2+, Cd2+, and Mn2+). The mandatory requirement for a hydrophobic chelator and transition metals, capable of binding two (or more) chelators simultaneously, was demonstrated. The potential generality of the mechanism presented derives from the observation that different combinations of [detergent : chelator : metal] are able to induce specific micellar clustering. The greater solubilization capacity of tethered-micelles in comparison with untethered micelles was demonstrated when the water insoluble aromatic molecule fluorenone (8 mM = 1.44 mg mL−1) and two highly lipophilic antibiotics: chloramphenicol (5 mM = 1.62 mg mL−1) and tetracycline (1.5 mM = 0.66 mg mL−1) were solubilized – only when the micelles were tethered.

Purification of His-Tagged Proteins with [Desthiobiotin−BSA−EDTA] Conjugates Exhibiting Resistance to EDTA

Two His-tagged proteins (His 6-P38 and His 6-Protein A) were purified by specific precipitation utilizing nonsoluble macrocomplexes composed of: BSA conjugates (modified with desthiobiotin-NHS and EDTA-dianhydride), tetrameric avidin, and Cu2+ ions. The generated pellets containing bound His-tagged proteins are washed with EDTA (25-100 mM) and then eluted in relatively high purity (> or =90%) devoid the macrocomplexes. Three different BSA conjugates were synthesized (DB-BSA-EDTA, DB-BSA-EDTA-A, DB-BSA-EDTA-B) and their adsorption capacities (3.8-6.4 micromol/g of BSA conjugate) as well as the recovery yields of His-tagged proteins obtained with them (44-84%) determined. The data demonstrate that capacity is dependent on the stochiometric ratio of modifying reagents (i.e., desthiobiotin-NHS and EDTA-dianhydride) used during the synthesis of the BSA conjugates. Copper ions were found to be significantly superior to Zn2+, Co2+, and Ni2+. BSA conjugates could be regenerated in moderate yields (74-83%) by incubating them at 88 degrees C in the presence of biotin (10 mM) at pH 7. The absence of resins leads to formation of small pellets (1-5 mg) and utilization of minute volumes of elution buffer (50-100 microL). Hence, concentrated preparations can be obtained, and a reconcentration step may be circumvented.

A general approach for antibody purification utilizing [protein A-catechol:Fe3+] macro-complexes

A general platform for antibody purification utilizing free nonimmobilized Protein A modified with the strong metal chelator catechol (ProA-CAT) and Fe3+ ions is presented. The mechanism of purification requires formation and precipitation of macro-complexes composed of [ProA-CAT:IgG:Fe3+]. Target IgGs are eluted directly from the precipitates (i.e. pellets) at pH 3 in high yields (71-80%) and high purity (>95%), without dissociating the [ProA-CAT:Fe3+] insoluble macro-complex.

Membrane protein crystallization in micelles conjugated by nucleoside base-pairing: A different concept.

The dearth of high quality, three dimensional crystals of membrane proteins, suitable for X-ray diffraction analysis, constitutes a serious barrier to progress in structural biology. To address this challenge, we have developed a new crystallization medium that relies on the conjugation of surfactant micelles via base-pairing of complementary hydrophobic nucleosides. Base-pairs formed at the interface between micelles bring them into proximity with each other; and when the conjugated micelles contain a membrane protein, crystal nucleation centers can be stabilized, thereby promoting crystal growth. Accordingly, two hydrophobic nucleoside derivatives - deoxyguanosine (G) and deoxycytidine (C), each covalently bonded to a 10 carbon chain were synthesized and added to an aqueous solution containing octyl β-d-thioglucopyranoside micelles. These hydrophobic nucleosides induced the formation of oil-rich globules after 2days incubation at 19°C or after a few hours in the presence of ammonium sulfate; however, phase separation was inhibited by 100mM GMP. The presence of the membrane protein bacteriorhodopsin in the conjugated - micellar dispersion resulted in the growth within the colorless globules of a variety of purple crystals, the color indicating a functional protein. On this basis, we suggest that conjugation of micelles via base-pair complementarity may provide significant assistance to the structural determination of integral membrane proteins.

Engineered-membranes: A novel concept for clustering of native lipid bilayers

A strategy for clustering of native lipid membranes is presented. It relies on the formation of complexes between hydrophobic chelators embedded within the lipid bilayer and metal cations in the aqueous phase, capable of binding two (or more) chelators simultaneously Fig. 1. We used this approach with purple membranes containing the light driven proton pump protein bacteriorhodopsin (bR) and showed that patches of purple membranes cluster into mm sized aggregates and that these are stable for months when incubated at 19°C in the dark. The strategy may be general since four different hydrophobic chelators (1,10-phenanthroline, bathophenanthroline, Phen-C10, and 8-hydroxyquinoline) and various divalent cations (Ni(2+), Zn(2+), Cd(2+), Mn(2+), and Cu(2+)) induced formation of membrane clusters. Moreover, the absolute requirement for a hydrophobic chelator and the appropriate metal cations was demonstrated with light and atomic force microscopy (AFM); the presence of the metal does not appear to affect the functional state of the protein. The potential utility of the approach as an alternative to assembled lipid bilayers is suggested.