Folding Proteins and Assembling Viruses. Insights in the Biosciences using Ultrasenstive Fluorescence Methods

by Don lamb (Dept. of Chemistry, Ludwig-Maximilians-Universitat Munchen)

Monday 30 Nov 2009, 11:00 → 12:00 Europe/Zurich

EPFL, Auditoire II du BSP

Heat Shock Proteins are involved in numerous processes in the cell including protein folding, prevention of protein aggregation and translocation of proteins across membranes. In this talk, I will summarize results from measurements on two types of heat shock proteins, GroEL, a Chaperonin and the class of HSP70 proteins. Using burst analysis with pulsed interleaved excitation, we have performed single-pair Förster resonance energy transfer (FRET) experiments to monitor the conformation of a protein substrate at different time points during the folding cycle of GroEL or the conformation of HSP70 upon interactions with nucleotides, co-chaperones and substrates. Many proteins require the assistance of molecular chaperones to fold rapidly and efficiently. Chaperone proteins are believed to aid protein folding by isolating the nascent polypeptide chain from deleterious interactions within the cell. Our results suggest a much more active involvement of GroEL in the folding of nascent proteins. HSP70 consists of two functional domains: the N-terminal ATPase domain where ATP and ADP can bind and the C-terminal substrate binding domain which can bind short peptides. Binding of ATP/ADP drives conformational changes in this protein though the nature of these changes remains elusive. We have investigated the conformation of HSP70 by cloning a number of mutants that allowed specific labeling of the different domains. These mutants make it possible to follow lid closure and interdomain separation in the presence of ATP or ADP as well as in the absence or presence of peptide substrates or co-chaperones. With the aid of single-particle tracking, we have investigated the assembly of HI-viruses. The main structural protein of HIV (GAG) has been fluorescently labeled with GFP. As a virus assembles, GAG proteins are delivered to the budding site. We have investigated the dynamics of the virus assembly process, visualizing whether GAG is delivered from the membrane or directly from the cytosol and determined the size of the budding sites. By monitoring the mobility of the budding sites, we have also been able to visualize virus release.