Protein Folding, Assembly and Degradation

Abstract

Students will carry out defined research projects related to the current research topics of the groups of Prof. Glockshuber and Prof. Weber-Ban. The topics include mechanistic studies on the assembly of adhesive pili from pathogenic bacteria, disulfide bond formation in the bacterial periplasm, ATP-dependent chaperone-protease complexes and formation of amyloid deposits in Alzheimer's disese.

Objective

The course should enable the students to understand and apply biophysical methods, in particular kinetic and spectroscopic methods, to unravel the mechanism of complex reactions of biological macromolecules and assemblies in a quantitative manner.

Content

The students will be tutored in their experimental work by doctoral or postdoctoral students from the Glockshuber or Weber-Ban group. In addition, the course includes specific lectures that provide the theoretical background for the experimental work, as well as excercises on the numeric evaluation of biophysical data, and literature work. Participation in one of the following projects will be possible: Projects of the Glockshuber group: - Purification, biophysical characterization and structure determiation of enzymes required for disulfide bond formation in the periplasm of Gram-negative bacteria. - Mechanistic studies on the assembly of type 1 pili from pathogenic Escherichia coli strains. In vitro reconstitution of pilus assembly from all purified components. Characterization of folding, stability and assembly behaviour of individual pilus subunits. - Identification of intermediates in the aggregation of the human Abeta peptide Experimental work on these projects involves - Molecular cloning, recombinant protein production in E. coli and protein purification - Protein crystallization - Thermodynamic and kinetic characterization of conformational changes in proteins and protein-ligand interactions by fluorescence and circular dischoism spectroscopy - Analysis of rapid reactions by stopped-flow fluorescence - Negative-stain electron microscopy - Light scattering Projects of the Weber-Ban group: - Generation and purification of site-directed variants of the E. coli ClpA/P protease and chaperone-proteasome complexes from other organisms, their biophysical characterization, including rapid kinetics by stopped-flow methods, ATPase activity measurtements, negative-stain electron microscopy and light scattering

Resources