Elucidation of the ATP7B N-Domain Mg2+-ATP Coordination Site and Its Allosteric Regulation

The diagnostic of orphan genetic disease is often a puzzling task as less attention is paid to the elucidation of the pathophysiology of these rare disorders at the molecular level. We present here a multidisciplinary approach using molecular modeling tools and surface plasmonic resonance to study the function of the ATP7B protein, which is impaired in the Wilson disease. Experimentally validated in silico models allow the elucidation in the Nucleotide binding domain (N-domain) of the Mg2+-ATP coordination site and answer to the controversial role of the Mg2+ ion in the nucleotide binding process. The analysis of protein motions revealed a substantial effect on a long flexible loop branched to the N-domain protein core. We demonstrated the capacity of the loop to disrupt the interaction between Mg2+-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process

Oxidative protein labeling in mass-spectrometry-based proteomics

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)–mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade

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-beta-D-maltoside (DDM), octyl beta-D-1-thioglucopyranoside (OTG), and n-octyl-beta-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

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-Cl 0, and 8-hydroxyquinoline) and various divalent cations (Ni2+, Zn2+, Cd2+, Mn2+, and Cu2+) 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. (C) 2012 Elsevier Inc. All rights reserved

Engineered-membranes and engineered-micelles as efficient tools for purification of halorhodopsin and bacteriorhodopsin

We describe two alternative and complementary purification methods for halorhodopsin and bacteriorhodopsin. The first relies on a unique form of detergent micelles which we have called engineered-micelles. These are specifically conjugated in the presence of [hydrophobic chelator: Fe2+] complexes and form detergent aggregates into which membrane proteins partition, but hydrophilic water-soluble proteins do not. The approach was tested on the membrane protein, bacteriorhodopsin (bR), with five non-ionic detergents (OG, OTG, NG, DM, and DDM), commonly used in purification and crystallization of membrane proteins, in combination with the commercially available bathophenanthroline or with one of the three synthesized phenanthroline derivatives (Phen-C10, Phen-C8 and Phen-C6). Our results show that bR is extracted efficiently (60-86%) and directly from its native membrane into diverse detergent aggregates with preservation of its native conformation, while 90-95% of an artificial contaminating background is excluded. For implementation of the second method, based on engineered-membranes, the use of detergents, which in some cases may produce protein denaturation, is not required at all. Protein-containing membranes are conjugated via the same hydrophobic [ chelator: metal ion] complexes but maintain the membrane protein in its native bilayer environment throughout the process. This method is demonstrated on the membrane protein halorhodopsin from Natronomonas pharaonis (phR) and leads to good recovery yields (74-89%) and removal of >85% of artificial background impurities while preserving the native state of phR. The detailed structure of the hydrophobic chelator used has been found to have a marked effect on the purity and yield of both methods