Using an innovative new imaging technique, researchers at Weill Cornell Medicine have revealed the inner workings of a family of light-sensitive molecules with unprecedented detail and speed. The work could inform new strategies in the burgeoning field of optogenetics, which uses pulses of light to alter the activity of individual neurons and other cells.
Light-sensitive proteins drive many crucial processes in biology, ranging from photosynthesis to vision. Much of the scientific community’s understanding of these proteins comes from studies of bacteriorhodopsin, a protein responsible for photosynthesis in certain unicellular organisms. Researchers have previously solved the three-dimensional structure of bacteriorhodopsin and studied its activity in detail, but the limitations of available techniques left puzzling gaps in the resulting models.
The new to learn, published December 10 in nature communication, describes a technique the researchers developed called line-scanning high-speed atomic force microscopy, which captures the movements of bacteriorhodopsin in response to light on a millisecond scale.
“Solving protein structures has become quite easy,” said the senior author Dr. Simon Scheuring, Professor of Physiology and Biophysics in Anesthesiology at Weill Cornell Medicine. “But a current challenge is the assessment of the kinetics, which provides a dynamic understanding of the system.”
In particular, other methods that track the activity of single molecules work too slowly to show how the protein changes over short periods of time, as bacteriorhodopsin appears to do in response to light. dr Scheuring likens these techniques to a slow-shutter film camera, which can capture a fast-moving bird on one side of the screen and then the other, but cannot track it between those two points.
Previously, researchers have addressed this problem by handicapping the bird: by looking at different forms of bacteriorhodopsin. “To study the kinetics of bacteriorhodopsin, people used mutants that were slower,” said the first author Dr. Alma Perez Perrino, a postdoctoral fellow in the lab of Dr. Scheuring. However, the slower variants do not represent the normal activity of the protein. To counteract this, Dr. Perez Perrino and her colleagues used high-speed line-scanning atomic force microscopy that sacrifices some image details for a much faster frame rate, such as B. blurrier images of the bird to follow it all the way up the screen.
“We track the protein every 1.6 milliseconds, which allowed us to study the speed of wild-type bacteriorhodopsin,” said Dr. Perez Perrino.
In response to light, bacteriorhodopsin switches between open and closed states. Using their faster imaging technique, the researchers discovered that the transition to the open state and the duration of the open state always occur at the same rate, but the molecule stays in the closed state longer as the light intensity decreases.
Optogenetics smuggle genes for light-sensitive molecules into neurons or other cells and can thus change the behavior of the cells with light pulses. This work has revolutionized neuroscience and also holds potential for the treatment of neurological disorders. The more researchers know about light-sensitive proteins, the further they can push optogenetics. “After all, you want to switch on a process, then get the maximum out of it and be able to switch it off again immediately,” says Dr. Scheuring. “It is therefore very important to know the kinetics of the molecules for this switching.”
Video of a kymogram of wild-type bacteriorhodopsin responding to light