Protein turnover studies using single-molecule microscopy in functional bacterial flagellar motors of live cells to assess molecular complex stability

Most of the vital activities in living cells are carried out by proteins, so small that 1 billion could fit on a full-stop. Many of these processes require collections of proteins to assemble together into functional biological machines, several of which are to be found in the cell membrane. The flagellar motor of bacteria is such a machine, and is an ideal example to study since we have developed several techniques which allow us to observe the individual proteins as well as monitoring the machine's functional state. We can see where the proteins are located in the cell by tagging them with a small marker which glows when we shine the right colour of light on it, and by measuring the total brightness of this glow in the motor relative to the brightness of just a single tag we can estimate how many of the tagged proteins are present. The functional state of the machine is given by the motor speed. This can be monitored both by tethering the whole cell to a surface via one of its flagellar filaments and watching the cell rotate about this point, or by sticking the cell down to a surface and detecting the position of a small tag placed on a free flagellar filament. The bacterial flagellar motor is an ingenious device which spans the cell membrane and converts some of the electrical and chemical energy of ions into rotary mechanical motion of the motor, thereby bringing about rotation of an attached helical filament and allowing the cell to swim through its liquid surroundings. Previously we discovered that individual components of the motor are rapidly replaced, which may mean that biological components wear out and need replacing just as they do in man-made machines. There is now evidence that the some of these components may be held in place by the presence of the very ions which fuel the rotation. To fully test whether proteins do indeed degrade, how the presence of the surrounding ions affects the stability of the machine and how functioning of the machine affects how and when parts are replaced, we propose to monitor the movement and number of several motor components to a precision of single protein molecules whilst simultaneously monitoring the rotation speed of the motor in varieties of modified bacterial cells in which we can precisely and dynamically control the magnitude of the ion gradient across the cell membrane.

Approximately a third of all proteins are integrated in biological membranes, many as components of multimeric complexes, performing vital and diverse cellular functions. Some of the most essential processes are carried out by such assemblies in the cell membrane. The bacterial flagellar motor is such a machine, a large membrane spanning complex which converts electrochemical energy from a transmembrane ion-gradient of either sodium ions or protons to mechanical rotation energy, ultimately resulting in whole cell motility of the bacterium by swimming through liquid media. It is an ideal candidate for investigating a single protein complex in vivo; several techniques exist which permit the motor rotation speed to be monitored in a single living cell, which provides an instantaneous and precise indicator of machine function. Using GFP fusions to protein components of the motor we have developed advanced optical techniques to locate a motor protein dynamically in a single living cell in real time to an accuracy of a few nanometres, with stoichiometry and turnover estimated to a precision of single molecules using high-contrast microscopy such as total-internal-reflection fluorescence (TIRF). We propose to use these techniques to monitor changes to dynamic turnover and stoichiometry in response to changes in protein expression levels, transmembrane ion-motive force, motor rotation speed and protein degradation. We will vary ion-motive force either by varying pH and extra-cellular sodium ion concentration with a chimeric sodium-driven motor or by balancing protein influx against protein efflux through the light-driven pump proteorhodopsin or photosynthetic electron trasport with the native proton-driven motor. We will monitor GFP fusions to the stator (MotA and MotB), rotor (FliG) and switch-complex (FliM) proteins with single-molecule fluorescence microscopy, and motor speed with laser interferometry.