Jean-Luc Popot

An atypical haem in the cytochrome b6f complex

Photosystems I and II (PSI and II) are reaction centres that capture light energy in order to drive oxygenic photosynthesis; however, they can only do so by interacting with the multisubunit cytochrome b6f complex. This complex receives electrons from PSII and passes them to PSI, pumping protons across the membrane and powering the Q-cycle. Unlike the mitochondrial and bacterial homologue cytochrome bc1, cytochrome b6f can switch to a cyclic mode of electron transfer around PSI using an unknown pathway. Here we present the X-ray structure at 3.1 Å of cytochrome b6f from the alga Chlamydomonas reinhardtii. The structure bears similarities to cytochrome bc1 but also exhibits some unique features, such as binding chlorophyll, β-carotene and an unexpected haem sharing a quinone site. This haem is atypical as it is covalently bound by one thioether linkage and has no axial amino acid ligand. This haem may be the missing link in oxygenic photosynthesis.

Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose–neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Membrane protein folding: beyond the two stage model

The folding of α‐helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable α‐helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process.

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.

Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1

The respiratory chain in the inner mitochondrial membrane contains three large multi‐enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19‐Å 3D map of the 1.7‐MDa amphipol‐solubilized supercomplex I1III2IV1 from bovine heart obtained by single‐particle electron cryo‐microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X‐ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol‐binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.

Amphipols, Nanodiscs, and Fluorinated Surfactants. Three Nonconventional Approaches to Studying Membrane Proteins in Aqueous solutions

Membrane proteins (MPs) are usually handled in aqueous solutions as protein/detergent complexes. Detergents, however, tend to be inactivating. This situation has prompted the design of alternative surfactants that can be substituted for detergents once target proteins have been extracted from biological membranes and that keep them soluble in aqueous buffers while stabilizing them. The present review focuses on three such systems: Amphipols (APols) are amphipathic polymers that adsorb onto the hydrophobic transmembrane surface of MPs; nanodiscs (NDs) are small patches of lipid bilayer whose rim is stabilized by amphipathic proteins; fluorinated surfactants (FSs) resemble detergents but interfere less than detergents do with stabilizing protein/protein and protein/lipid interactions. The structure and properties of each of these three systems are described, as well as those of the complexes they form with MPs. Their respective usefulness, constraints, and prospects for functional and structural studies of MPs are discussed.

Amphipols: Polymeric surfactants for membrane biology research

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.

Major myelin proteolipid the 4-α-helix topology

SummarySeveral conflicting models have been proposed for the membrane arrangement of the major myelin proteolipid (PLP). We have compared features of the sequence of PLP with those of other eukaryotic integral membrane proteins, with the view of identifying the most likely transmembrane topology. A new, simple model is suggested, which features four hydrophobic α-helices spanning the whole thickness of the lipid bilayer. Its orientation may be such that both the N-and C-termini face the cytosol. None of the biochemical, biophysical or immunological experiments hitherto reported provides incontrovertible evidence against the model. The effect or absence thereof of various PLP mutations is discussed in the frame, of the proposed 4-helix topology.

Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus.

Hearing impairment affects about 1 in 1,000 children at birth. Approximately 70 loci implicated in non-syndromic forms of deafness have been reported in humans and 24 causative genes have been identified (see also http://www.uia.ac.be/dnalab/hhh). We report a mouse transcript, isolated by a candidate deafness gene approach, that is expressed almost exclusively in the inner ear. Genomic analysis shows that the human ortholog STRC (so called owing to the name we have given its protein—stereocilin), which is located on chromosome 15q15, contains 29 exons encompassing approximately 19 kb. STRC is tandemly duplicated, with the coding sequence of the second copy interrupted by a stop codon in exon 20. We have identified two frameshift mutations and a large deletion in the copy containing 29 coding exons in two families affected by autosomal recessive non-syndromal sensorineural deafness linked to the DFNB16 locus. Stereocilin is made up of 1,809 amino acids, and contains a putative signal petide and several hydrophobic segments. Using immunohistolabeling, we demonstrate that, in the mouse inner ear, stereocilin is expressed only in the sensory hair cells and is associated with the stereocilia, the stiff microvilli forming the structure for mechanoreception of sound stimulation.

Membrane protein-surfactant complexes

Transmembrane proteins expose to the surrounding membrane a belt of mainly hydrophobic amino acid residues, which makes them insoluble in water. Solubilizing them and handling them in vitro generally relies on the use of dissociating surfactants (detergents). Exposing membrane proteins to detergents, however, adversely affects their stability, which is a major hindrance in their study. After briefly recalling relevant aspects of membrane protein structure, the modus operandi of detergents and the problems they raise, we describe alternative approaches such as insertion into bicelles or lipid cubic phases, or association with non-detergent amphiphiles such as peptitergents, hemifluorinated surfactants and amphipols. These novel supramolecular assemblies offer a fascinating playground for collaborative studies between organic chemists, physical chemists and biologists, and they have spurred imaginative works in each of these fields.