Single molecule studies on the Sec-translocase

PhD ceremony: Mr. I. Küsters, 11.00 uur, Academiegebouw, Broerstraat 5, Groningen

Dissertation: Single molecule studies on the Sec-translocase

Promotor(s): prof. A.J.M. Driessen

Faculty: Mathematics and Natural Sciences

In his thesis Ilja Küsters has determined the quaternary structure of SecA and SecYEG in a native like environment that sustains the activity of the entire translocation system. To this end, he has developed new fluorescence based methods and applied them to monitor the oligomeric state of proteins within or associated with the lipid bilayer. Dual-color fluorescence burst analysis is a new method that enables investigation of protein-protein interactions in highly diluted solutions

Protein secretion in bacteria is mediated by a multi-protein machinery termed the translocon. The SecA motor protein translocates secretory protein through the membrane channel SecYEG using ATP as an energy source. Albeit a multitude of biochemical and biophysical studies on the oligomeric state of the subunits of the translocon have been conducted the topic remains controversial. This is mainly due to the challenge to determine oligomeric states of proteins at or in the membrane interface.

Purification, site specific fluorescent labeling and functional reconstitution of the translocon allowed the assessment of protein translocation in vitro. In order to monitor ligand-membrane receptor interactions in equilibrium and with single molecule sensitivity, a recently developed method for fluorescence detection was modified by Küsters. Using this method he has found that the motor-protein SecA binds as a dimer (two SecA molecules bound to each other) to the membrane pore SecYEG. And also he found that the protein conducting membrane channel SecYEG ismonomeric (one SecYEG molecule alone) in the membrane.

To address kinetic parameters of the translocation reaction, new immobilization techniques for membrane vesicles that sustain the activity of (membrane) proteins are developed by him. Hydrogels formed from organic gelators (made by Biomade) proved to be efficient in immobilizing membrane vesicles and allow observation of biological processes on single vesicles.

Furthermore, he has applied a modified fluorescence detection methodto measure protein translocation activity with single molecule sensitivity. Using these technologies, future studies will reveal mechanistic details of protein translocation at the single molecule level that are difficult to study with the typical ensemble of proteins in solution.