Coupling Optical and Electrical Measurements in Artificial Membranes: Lateral Diffusion of Lipids and Channel Forming Peptides in Planar Bilayers

Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K(+) channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules

Protein Folding In Vivo

Proteins are composed of linear chains of amino acids. Upon synthesis in the cell, most proteins must rapidly acquire a specific three-dimensional structure, a process known as folding, before they can perform their various biological func- tions. Productive folding is often competed by aggregation, owing to the high macromolecular crowding of the cellular environment. Moreover, the process of translation increases the danger of misfolding, as incomplete nascent polypeptides are not yet able to fold into stable structures in many cases. To avoid these off-pathway reactions, a class of proteins called molecular chaperones has evolved in all organisms. They interact with nascent or stress-denatured polypeptides, prevent their aggregation and assist in folding and assem- bly processes, often in an ATP-regulated manner. These functions are especially important in condi- tions of cell stress, and their failure is linked with the manifestation of numerous age-dependent degenerative disease

Rescue of the neuroblastoma mutant of the human nucleoside diphosphate kinase A/nm23-H1 by the natural osmolyte trimethylamine-N-oxide

International audienc

Crystal structures of S120G mutant and wild type of human nucleoside diphosphate kinase A in complex with ADP

International audienc

The structure of E.coli nucleoside diphosphate kinase broadens the diversity in the quaternary architecture of NDKs.

International audienc

GroEL/ES Chaperonin Modulates the Mechanism and Accelerates the Rate of TIM-Barrel Domain Folding

The GroEL/ES chaperonin system functions as a protein folding cage. Many obligate substrates of GroEL share the (beta alpha)(8) TIM-barrel fold, but how the chaperonin promotes folding of these proteins is not known. Here, we analyzed the folding of DapA at peptide resolution using hydrogen/deuterium exchange and mass spectrometry. During spontaneous folding, all elements of the DapA TIM barrel acquire structure simultaneously in a process associated with a long search time. In contrast, GroEL/ES accelerates folding more than 30-fold by catalyzing segmental structure formation in the TIM barrel. Segmental structure formation is also observed during the fast spontaneous folding of a structural homolog of DapA from a bacterium that lacks GroEL/ES. Thus, chaperonin independence correlates with folding properties otherwise enforced by protein confinement in the GroEL/ES cage. We suggest that folding catalysis by GroEL/ES is required by a set of proteins to reach native state at a biologically relevant time scale, avoiding aggregation or degradation

Aggregation of the neuroblastoma-associated mutant (S120G) of the nucleoside diphosphate kinase A-NM23-H1 into mayloid fibrils

International audienc