Stephen Sligar's laboratory has a diverse range of research topics, investigating aspects of chemistry and biology from the nanoscale on up. Through all the diversity, however, the lab has found a unifying theme in a research method Sligar developed in 1999.
Sligar, a Beckman Institute faculty member in the 3-D Micro and Nanosystems group, discovered a system for creating a self-assembled nanoscale lipid bi-layer termed "Nanodiscs" that gives researchers a way to study and manipulate molecular structures like proteins in ways no one had done before.
"The Nanodisc system has been such an enabling technology for us to look at that, in fact, it's become a common theme in our laboratory," Sligar said. "Even though we work on very different systems - blood coagulation, drug metabolism, receptors and signaling pathways - all of those have actually been enabled by this Nanodisc technology. Now we're seeing what used to be a very eclectic lab all using this technology in a fundamental way."
The Nanodisc technology was a key part of two current projects in which Sligar and his collaborators have reported important new findings on blood clotting and on an enzyme heavily involved in human drug metabolism. One paper authored by Sligar, Beckman postdoctoral researcher Ilia Denisov and others, focuses on an enzyme called cytochrome P450 that plays a crucial role in various metabolic processes, including drug metabolism. The other by Sligar, Biochemistry Professor James Morrissey, and graduate research assistants Andrew Shaw and Vincent Pureza provides new insight into how blood clotting is activated in the body.
- Stephen Sligar
"We've been able to use a simple process to make Nanodiscs in which we have incorporated a variety of membrane proteins, including P450s, or the blood coagulating factors, or other systems," Sligar said. "So it's been a powerful tool for a large number of membrane proteins."
Sligar noted that the Nanodisc technology has so far proven successful with every type of membrane protein for which it has been applied. The research results could play major roles in advancing drug therapies, especially in understanding drug-drug interactions, and in creating new methods for preventing blood loss. Sligar describes the Nanodisc creation technique as a simple, yet almost magical, process.
Using 130 to 160 phospholipids (organic compounds found in biological membranes) to create a structure, researchers then surround the structure with a protein membrane scaffold belt. The process then calls for a detergent that, once removed, miraculously allows the Nanodiscs to self-assemble into hockey puck-like, bi-layer membrane structures. Depending on the method used, membrane proteins can be incorporated into the Nanodisc in order to stabilize and study them, or the Nanodisc can be used to "recruit" enzymes that will bind to it for initiating processes like blood clotting.
Sligar said P450 plays a role in activating dangerous compounds like carcinogens, as well as metabolizing compounds such as ingested hydrocarbons and drugs or the compounds in antibiotics. Understanding the workings of P450 in the liver may prove to be the most useful benefit of the research because of its importance in developing new therapeutic compounds.
"I am very proud of this recent paper in the Journal of Biological Chemistry because it's a major step in understanding human health," Sligar said. "One of the big problems that is facing the pharmaceutical industry is that many drugs you might be taking may be interacting with other ingested drugs, which may have critical effects on subsequent metabolism.
"So what we've discovered is the mechanism by which this enzyme in your liver interacts with a variety of substrates and what controls the interaction with these various compounds, these various drugs. It allows us now to predict what kinds of drug-drug interactions might be beneficial or deleterious to the organism."
The research on blood clotting could lead to applications such as band-aids and bandages that are based on the Nanodisc technology.
"We can now, in a very small, stable environment, recruit the enzymes that are involved in this blood coagulation process," Sligar said. "We can put these Nanodiscs containing these factors, these coagulation factors, onto a surface.
"In the case of the blood coagulating enzymes, it's the enzymes that are recruited to the membrane surface to bind to a scaffold that's incorporated in the membrane bi-layer called tissue factor, and that initiates this cascade process that coagulates and stops bleeding."
Sligar said the Armed Forces have shown interest in the research because of its potential for use in trauma situations where there is a need to quickly control bleeding.
One of the most important and unique capabilities Nanodiscs offer researchers are that they are water soluble, an excellent attribute for working with enzymes like P450 and understanding their role in metabolism.
"What's made it difficult to study proteins and things that are associated with proteins has been the inability to take those entities out of the normal membrane and then put them in something that would allow you to then study those molecules," Sligar said. "So both of these experiments use these Nanodiscs as water, aqueous, solutions, in this discoidal, bi-layer structure where we've incorporated the target protein."
Sligar said the blood clotting project was a very interdisciplinary collaboration that allowed him to work with people he normally wouldn't as part of his research like Animal Sciences Professor Lawrence Schook.
"One of the things that has made this campus great is that there is a very open collaborative environment," Sligar said. "That doesn't happen at a lot of universities. It's a very open place. It's absolutely trivial on this campus to go talk to somebody in agriculture or engineering, and so on, and have an exciting conversation. That's not true on a lot of campuses and I think that's one of our big strengths here."