Super-strong molecular tool becomes even stronger
Researchers in the department have engineered changes in a widely-used molecular detection and targeting tool that will enable it to be used in an even greater range of biological and clinical applications.
Crash-testing traptavidin with molecular motors. Traptavidin (red with mutated amino acids in blue) bound to biotinylated DNA, with FtsK (yellow and black) approaching at 5kb/s.
University Lecturer Dr Mark Howarth and D.Phil. student Claire Chivers carried out the work with Professor David Sherratt and Dr Estelle Crozat in the department, and collaborators at the University of Miami. They have recently published their research on the engineered tool and its application to study the strength of a bacterial molecular motor (1).
The tool is a 2-component system made up of a small molecule called biotin and a protein called streptavidin which bind specifically and tightly to each other. Researchers exploit this strong interaction, using the molecular pairing as a way of purifying proteins, imaging molecules, in nano-assembly and clinical trials for cancer.
But the association between the two partners is not as strong as it needs to be for some key applications and the two can fall apart under certain conditions. "The interaction fails at high temperatures, in the presence of sheer forces and also at low pH," explains Claire Chivers.
Dr Howarth's group is interested in developing new chemical and biological approaches for imaging molecules and for diagnosis. He wanted to see whether he could widen the applications of the streptavidin-biotin system.
"We ended up making around 30 to 40 mutants of streptavidin, which were nearly all worse. This one jumped out."
"We were looking at trying to push this interaction even further to decrease this dissociation that's seen. We didn't expect that it would be possible to find something with stronger binding as we thought the interaction was already fully optimised," he says.
"We looked at the structure and tried to rationalise the choice of mutations to make," adds Claire. "We ended up making around 30 to 40 mutants of streptavidin, which were nearly all worse. This one jumped out."
Named traptavidin, the mutant protein falls apart from biotin at a rate which is more than ten times slower than that of the wild type streptavidin. It is a little slower at binding to biotin in the first place - but it gets there eventually. Binding of traptavidin to biotin is also more stable at high temperatures than binding of its streptavidin counterpart.
With this stable mutant in hand, the group decided to use it to probe the function of a bacterial molecular motor, FtsK, a protein studied by the Sherratt lab.
"FtsK is involved in bacterial chromosome segregation," says Dr Howarth. "When bacteria divide, they have to make sure that one chromosome gets into each daughter cell. FtsK sits at the junction between the cells and pumps the DNA so that there is one chromosome in each site. It's one of the fastest known molecular motors and moves the DNA at 5 kilobases per second, really very fast, and it's also somehow very powerful."
Its power helps it tackle the numerous obstacles - DNA-binding proteins and enzymes - that line its path when it passes along the DNA carrying out its job. The group wanted to test just how powerful it is using the streptavidin/biotin tool.
Dr Howarth explains: "We thought we'd give it a really strong challenge and see if it can push off streptavidin-biotin stuck on the DNA. What we found is that FtsK pushes it off really easily, in a few seconds."
The motor had less luck when trying to force its way through a traptavidin/biotin block. "When we put our mutant on, FtsK couldn't get it off," says Dr Howarth.
This is the first demonstration that in vivo FtsK should have no difficulty in displacing even strongly attached DNA-binding proteins. It also shows that traptavidin and weaker streptavidin mutants that are available can be used to calibrate the force that FtsK and other motors have to fight against.
"There are still a lot of questions about this motor that the system will contribute to investigating," says Dr Howarth (2).
Aside from this specific application, traptavidin's enhanced stability may make it more popular than streptavidin for point-of-care diagnostics and for single molecule DNA sequencing. Dr Howarth is keen to make trapavidin widely available. "We've got a patent, and a few companies are already planning to try it out. I would expect that it will be valuable in a wide range of applications."
For details of additional recent news from Dr Mark Howarth's lab, with