Protein folding and translocation : single-molecule investigations

Doctoral Thesis

This thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a protein that was being unfolded and refolded using our optical tweezers setup. Our measurements clearly showed that in the presence of SecB, an unfolded protein could not refold. Molecular dynamics simulations were used to successfully explain features that were observed in our unfolding experiments. Our approach enables studies on other chaperones, as well. Next, we aimed to study translocation of single proteins through membranes by the E. coli Sec translocase. We modified an often-used model protein for our experiment. Different used experimental strategies are presented. Future experiments should enable measurements on the translocation of a single protein. The last study was on the packaging of double-stranded DNA by a single bacteriophage phi29. We aimed to study the effect of...Show moreThis thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a protein that was being unfolded and refolded using our optical tweezers setup. Our measurements clearly showed that in the presence of SecB, an unfolded protein could not refold. Molecular dynamics simulations were used to successfully explain features that were observed in our unfolding experiments. Our approach enables studies on other chaperones, as well. Next, we aimed to study translocation of single proteins through membranes by the E. coli Sec translocase. We modified an often-used model protein for our experiment. Different used experimental strategies are presented. Future experiments should enable measurements on the translocation of a single protein. The last study was on the packaging of double-stranded DNA by a single bacteriophage phi29. We aimed to study the effect of multivalent cations on the negatively-charged, tightly-packed DNA inside the bacteriophage capsid and in that way on the speed of the packaging process. A special DNA molecule was constructed and used in a number of successful packaging experiments. Future experiments should show the effect of cations on the packaging rate.Show less

Protein translocation

Protein folding

Chaperones

Optical tweezers

Single-molecule biophysics