One of the major scientific advances of the late 20th and early 21st century was the ability to see individual protein molecules in real time using single-molecule fluorescence microscopy. In his postdoctoral research from 2006-2010, Dr. Knight pioneered the use of single-molecule fluorescence microscopy to observe individual fluorescent-tagged proteins binding to the surface of supported lipid bilayer membranes and diffusing in the 2-dimensional lipid bilayer in real time. He and his collaborators demonstrated that the diffusion constant of a membrane-bound protein is inversely proportional to its number of tightly bound lipids, and can even be used to measure whether a protein is a monomer or a dimer in real time.
Diffusion of membrane-bound proteins scales inversely with the number of bound lipids. Top: schematic diagram of monomer (1PH), dimer (2PH), and trimer (3PH) constructs used in this study. All are labeled with a single fluorophore on the N-terminus (hatched box). Middle: Representative images from single-molecule tracking movies. Each bright spot is a single fluorescent molecule. Bottom: Diffusion constants of the mobile states decrease proportionally to the oligomeric state. Modified from Knight et al, Biophys. J. 2010, 99, 2879-2887.
The Knight lab has used this single-molecule diffusion technique to study C2 domain proteins, including the tandem C2 domains of synaptotagmin-7. In our 2012 Biochemistry paper, we used single-molecule diffusion to demonstrate that properly purified Syt-7 C2A domains remain monomeric when bound to planar supported lipid bilayers, even at micromolar concentrations. In 2014, we used single-molecule diffusion along with kinetic measurements to demonstrate that the C2 domains of Syt-7 bind to simple lipid membranes as independent modules.
Snapshot from a video of individual synaptotagmin-7 C2 domains diffusing laterally on a supported lipid bilayer. Each bright spot is one protein molecule. From Vasquez et al, Biochemistry 2014, 53, 7904-7913.
The Knight lab continues to be interested in advancing single-molecule measurements of membrane-bound proteins. Can this method reveal protein-lipid interactions that other techniques don't? What can it tell us about oligomer formation and aggregation of membrane-bound proteins? Can we use specially developed substrate systems to study single protein molecules binding to curved membrane geometries? How do rate constants for membrane binding and release measured with single molecules compare to ensemble methods?
Single-molecule microscopy is technically challenging, both in doing the experiments and in analyzing the data. But these methods is fully within the reach of even undergraduate researchers in the Knight lab – the first author on our 2014 Syt-7 paper, Joseph Vasquez, was an undergraduate. Furthermore, Dr. Knight has developed and implemented single-molecule fluorescence experiments into an undergraduate class, CHEM 4548 Physical Biochemistry Laboratory. Students complete these experiments and analysis successfully every semester. Email Dr. Knight to find out more!