An interdisciplinary team of researchers at the University of Illinois’ Institute for Genomics Biology has developed chimeric polypeptides that it claims are useful as redox-sensitive linkers when used with Förster resonance energy transfer, or FRET.
The scientists placed α-helical linkers of different lengths that contained redox switches between a FRET pair of enhanced cyan fluorescent protein and enhanced yellow fluorescent protein motifs that were sensitive to the environmental redox state.
They tested these constructs for efficacy using the (ratio)A method and found that the cyan/yellow construct carrying one of the redox linkers, RL5, exhibited a 92 percent increase in FRET efficiency from its reduced to its oxidized state.
The research, which combines cell biology and engineering, appears in the February issue of Experimental Biology and Medicine.
“The key was to develop the biosensor between the donor-acceptor,” said Paul Kenis, an associate professor of chemical and biomolecular engineering at the University of Illinois at Urbana-Champaign, and a co-author of the paper. “Basically Vladimir Kolossov and a couple of colleagues created a variety of these linkers and were able to identify one that works really well. That was key, because if that piece is very sensitive to change in a redox environment, it will bend from an α-helix formation to a clamped coil, and the donor and acceptor will be close enough to exchange excitation energy.”
Kolossov, a visiting assistant professor of animal sciences at the school, spoke with CBA News recently about the work and how it fits into technology that the team is developing to study the intracellular redox state of cells.
You said that you had another publication coming out shortly about a platform.
VK: We will talk about a platform on which we seed cells, and by applying a potential to that surface, we can drive the cells to either become more oxidized or more reduced. There is a direct correlation between the redox state of a cell and the cell cycle events leading to proliferation.
That is where we eventually want to go, to look at cell biology itself. The present paper discusses the linker, one of the crucial pieces that we will need for that.
To what publication do you plan to submit your work, and when do you plan to submit it?
VK: I hope within the next several weeks. Then it will take the usual several months for review, et cetera, before it can be published. That paper will not include any of the biosensor work.
A third future paper, on which we are starting work now, will hopefully combine the two. This is at least six months in the future, however.
How would this technology be applicable to drug discovery? Could it be used to look at the effect of candidate compounds on the cellular redox state?
VK: That is an interesting idea. Certain drugs have been known to impact specific processes in cell biology. Whether they have a profound effect on the redox state is a real question, because most drugs have not been characterized in that way.
It is difficult to say at this point if this may be a tool for drug discovery. That is a very speculative claim at this point.
Are these constructs and this platform something that you plan to commercialize?
VK: Right now we do not have any specific plans. We are currently focused on pursuing the overall goal of our research grant, which is looking at the role of redox states in cell cycle events, which is directly related to diseases such as cancer.
In cancer, as we know, excessive proliferation of cells is the problem. But how does that happen? Is the redox cycle getting messed up and then the cell gets diseased as a result? Or is the cell getting diseased in another way first, which in turn messes up the redox cycle? So it is a question of cause and consequence.
It is very difficult to monitor the redox state of the cell, so it has not been studied often with respect to its role in disease. There are all kinds of reports that indicate it may be important, but it has not been studied very much.
This is partly due to a lack of tools. This is where the engineering approach comes in.