Jean-Pierre Duneau

Characterization of the motion of membrane proteins using high-speed atomic force microscopy

For cells to function properly, membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein-protein and protein-lipid interactions. However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy. We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein-protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.

Single-spanning transmembrane domains in cell growth and cell-cell interactions More than meets the eye?

As a whole, integral membrane proteins represent about one third of sequenced genomes, and more than 50% of currently available drugs target membrane proteins, often cell surface receptors. Some membrane protein classes, with a defined number of transmembrane (TM) helices, are receiving much attention because of their great functional and pharmacological importance, such as G protein-coupled receptors possessing 7 TM segments. Although they represent roughly half of all membrane proteins, bitopic proteins (with only 1 TM helix) have so far been less well characterized. Though they include many essential families of receptors, such as adhesion molecules and receptor tyrosine kinases, many of which are excellent targets for biopharmaceuticals (peptides, antibodies, et al.). A growing body of evidence suggests a major role for interactions between TM domains of these receptors in signaling, through homo and heteromeric associations, conformational changes, assembly of signaling platforms, etc. Significantly, mutations within single domains are frequent in human disease, such as cancer or developmental disorders. This review attempts to give an overview of current knowledge about these interactions, from structural data to therapeutic perspectives, focusing on bitopic proteins involved in cell signaling.

Dissecting membrane protein architecture: An annotation of structural complexity.

α‐Helical membrane proteins exist in an anisotropic environment which strongly influences their folding, stability, and architecture, which is far more complex than a simple bundle of transmembrane helices, notably due to helix deformations, prosthetic groups and extramembrane structures. However, the role and the distribution of such heterogeneity in the supra molecular organization of membrane proteins remains poorly investigated. Using a nonredundant subset of α‐helical membrane proteins, we have annotated and analyze the statistics of several types of new elements such as incomplete helices, intramembrane loops, helical extensions of helical transmembrane domains, extracellular loops, and helices lying parallel to the membrane surface. The relevance of the annotation scheme was studied using residue composition, statistics, physical chemistry, and symmetry of their distribution in relation to the immediate membrane environment. Calculation of hydrophobicity using different scales show that different structural elements appear to have affinities coherent with their position in the membrane. Examination of the annotation scheme suggests that there is considerable information content in the amino acid compositions of the different elements suggesting that it might be useful for structural prediction. More importantly, the proposed annotation will help to decipher the complex hierarchy of interactions involved in membrane protein architecture.

Lateral organization of biological membranes

The lateral organization of biological membranes is of great importance in many biological processes, both for the formation of specific structures such as super-complexes and for function as observed in signal transduction systems. Over the last years, AFM studies, particularly of bacterial photosynthetic membranes, have revealed that certain proteins are able to segregate into functional domains with a specific organization. Furthermore, the extended non-random nature of the organization has been suggested to be important for the energy and redox transport properties of these specialized membranes. In the work reported here, using a coarse-grained Monte Carlo approach, we have investigated the nature of interaction potentials able to drive the formation and segregation of specialized membrane domains from the rest of the membrane and furthermore how the internal organization of the segregated domains can be modulated by the interaction potentials. These simulations show that long-range interactions are necessary to allow formation of membrane domains of realistic structure. We suggest that such possibly non-specific interactions may be of great importance in the lateral organization of biological membranes in general and in photosynthetic systems in particular. Finally, we consider the possible molecular origins of such interactions and suggest a fundamental role for lipid-mediated interactions in driving the formation of specialized photosynthetic membrane domains. We call these lipid-mediated interactions a ‘lipophobic effect.’

Structure of a protein–detergent complex: the balance between detergent cohesion and binding

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein–detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorinxa0A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.

Lipid perturbation by membrane proteins and the lipophobic effect

Understanding how membrane proteins interact with their environment is fundamental to the understanding of their structure, function and interactions. We have performed coarse-grained molecular dynamics simulations on a series of membrane proteins in a membrane environment to examine the perturbations of the lipids by the presence of protein. We analyze these perturbations in terms of elastic membrane deformations and local lipid protein interactions. However these two factors are insufficient to describe the variety of effects that we observe and the changes caused by membranes proteins to the structure and dynamics of their lipid environment. Other factors that change the conformation available to lipid molecules are evident and are able to modify lipid structure far from the protein surface, and thus mediate long-range interactions between membrane proteins. We suggest that these multiple modifications to lipid behavior are responsible, at the molecular level, for the lipophobic effect we have proposed to account for our observations of membrane protein organization.

Tryptophan-mediated Dimerization of the TssL Transmembrane Anchor Is Required for Type VI Secretion System Activity

The type VI secretion system (T6SS) is a multiprotein complex used by bacteria to deliver effectors into target cells. The T6SS comprises a bacteriophage-like contractile tail structure anchored to the cell envelope by a membrane complex constituted of the TssJ outer-membrane lipoprotein and the TssL and TssM inner-membrane proteins. TssJ establishes contact with the periplasmic domain of TssM whereas the transmembrane segments of TssM and its cytoplasmic domain interact with TssL. TssL protrudes in the cytoplasm but is anchored by a C-terminal transmembrane helix (TMH). Here, we show that TssL TMH dimerization is required for the stability of the protein and for T6SS function. Using the TOXCAT assay and point mutations of the 23 residues of the TssL TMH, we identified Thr194 and Trp199 as necessary for TssL TMH dimerization. NMR hydrogen-deuterium exchange experiments demonstrated the existence of a dimer with the presence of Trp185 and Trp199 at the interface. A structural model based on molecular dynamic simulations shows that TssL TMH dimer formation involves π-π interactions resulting from the packing of the two Trp199 rings at the C-terminus and of the six aromatic rings of Tyr184, Trp185 and Trp188 at the N-terminus of the TMH.