Electro-enzymatic ATP regeneration coupled to biocatalytic phosphorylation reactions.

Adenosine-5-triphosphate (ATP) is the main energy vector in biological systems, thus its regeneration is an important issue for the application of many enzymes of interest in biocatalysis and synthetic biology. We have developed an electroenzymatic ATP regeneration system consisting in a gold electrode modified with a floating phospholipid bilayer that allows coupling the catalytic activity of two membrane-bound enzymes: NiFeSe hydrogenase from Desulfovibrio vulgaris and F1Fo-ATP synthase from Escherichia coli. Thus, H2 is used as a fuel for producing ATP. This electro-enzymatic assembly is studied as ATP regeneration system of phosphorylation reactions catalysed by kinases, such as hexokinase and NAD+-kinase for respectively producing glucose-6-phosphate and NADP+.

Quartz crystal microbalance and atomic force microscopy to characterize mimetic systems based on supported lipids bilayer.

Quartz Crystal Microbalance (QCM) with dissipation and Atomic Force Microscopy (AFM) are two characterization techniques that allow describing processes taking place at solid-liquid interfaces. Both are label-free and, when used in combination, provide kinetic, thermodynamic and structural information at the nanometer scale of events taking place at surfaces. Here we describe the basic operation principles of both techniques, addressing a non-specialized audience, and provide some examples of their use for describing biological events taking place at supported lipid bilayers (SLBs). The aim is to illustrate current strengths and limitations of the techniques and to show their potential as biophysical characterization techniques.

Study of Amyloid Fibers Using Atomic Force Microscopy.

Atomic force microscopy (AFM) provides high-resolution images of the topography of amyloid fibers adsorbed on surfaces. This information is very useful to study their molecular assembly under various conditions. This chapter describes the basic protocols required to deposit fibers on flat surfaces and discusses some of the practical issues required to operate a good commercial microscope setup to obtain appropriate high-resolution AFM topographic images of amyloid fibers.

Supported Lipid Bilayers (SLBs) to Study Amyloid-Lipid Membrane Interactions with Atomic Force Microscopy.

Supported lipid bilayers (SLBs) are model membrane systems that can be used to study the interaction between amyloid fibers and membranes with atomic force microscopy (AFM). This chapter describes the preparation of SLBs on mica that can then be used as a substrate for fiber absorption. AFM can then be used to study the topography of the lipid-protein surface to study the evolution of the fibers, as well as the modifications on the membrane induced by their presence.