Volume 4, Issue 25 February 2007
Biomolecules in Motion
Haw Yang hard at work devising methods to study the chemistry of single molecules. Credit: Michael Barnes
Proteins are the parts that make living engines run. They supply cells with energy, build muscle and bone, and catalyze countless other reactions that let the spark of life burn bright. To do their jobs, proteins must curl around substrate molecules, stretch to let their substrates go, travel around cells and assemble into work crews.
Studying how proteins function, however, can be a major pain. Just billionths of a meter in size, they're too small to observe under a microscope. So scientists have resorted to more indirect methods, like mixing thousands of proteins together with other chemicals and observing the results.
While such averaging experiments have their uses, says UC Berkeley chemistry professor Haw Yang, "we are missing a lot of the interesting action" going on in individual biomolecules.
Now Yang is developing a better way to study biomolecules in motion. "What we hope is to visualize chemistry one molecule at a time."
For this feat, Yang uses a technique called single-molecule microscopy. The method employs probes that fluoresce red or blue when excited by a photon of light. The blue one can acquire photons from an external light source, while the red one only accepts photons from its blue counterpart. For his experiments, Yang affixes one blue and one red probe to opposite ends of a study enzyme. When the enzyme is relaxed, and both probes are far apart, the assembly glows blue. When the enzyme closes, the blue probe can pass along its photon, and the assembly glows red.
Glowing probes help Yang analyze the movement of enzymes. When the enzyme is open, the assembly glows red (left); when closed, the assembly glows blue (right). The color fluctuations reveal both an enzyme's activity rate and the number of positions, or conformations, it can assume. Credit: Lucas Watkins
"We can actually watch in real time as biomolecules such as enzymes open and close," Yang says.
The color fluctuations allow Yang to calculate how fast enzymes move and their range of motion. The amount of time they spend in various postures even indicates how many conformational states they have.
Scientists have long believed that when an enzyme is empty, it gapes open like a hungry alligator, and that after it has caught its substrate, it remains closed until the reaction has been completed. Yang's single-molecule microscopy studies have turned this notion upside-down. "Even when it has substrate, it doesn't just bind the substrate tightly and stop moving. It's still flapping," he says.
This constant motion makes perfect sense, considering how fast enzymes operate; some can process a million substrate molecules per minute. "Like a door, it has to be able to swing even without me going in and out. Its motion has to be inherent and already present in order to respond very quickly," Yang says. Relative instability enables enzymes to release substrate efficiently after a reaction is completed.
At present, Yang's single-molecule microscope is focused on an enzyme from the gut bacterium Escherichia coli. By comparing the behavior of the wild-type enzyme against enzymes with known mutations, he hopes to work out the design principles behind enzyme evolution.
The next step is to study how molecules work while inside a cell. To that end, Yang and his research group have invented a means to track the zigs and zags of a single molecule in three dimensions.
Yang and his group have developed a device capable of tracking the three-dimensional movements of a single nanometers-scale particle in solution. Above: an automated microscope keeps the target particle (encircled in red) in sharp focus as a neighboring particle (green arrow) drifts by. Below: The trajectory of the molecule plotted over time. Credit: Shan Xu
The system works much like the "missile lock" function on a fighter jet, though on a far smaller scale. Using a joystick connected to a powerful microscope and camera, Yang can target a gold nanoparticle, and the system will automatically keeps it in focus. On video, the target particle remains bright and sharp at the center of the screen, while other molecules drift in and out of focus like snowflakes in a storm. At present, Yang can record the three-dimensional location of a single 80-nanometer particle in water every 20 microseconds.
Meanwhile, a spectrophotometer records the light wavelengths refracted off the target as it rotates. This information allows Yang to calculate a particle's diffusion coefficient, which describes how easily it drifts in solution.
The device could help scientists better understand problems such as molecular self-assembly and protein translocation within a cell. For example, many different proteins are known to help manufacture proteins from RNA. But how and when they come together, and whether additional proteins are involved, remains unknown. The new focus-tracking microscope should provide new insights into this problem. Similarly, by observing the change in the diffusion coefficient that occurs when a protein lands on a strand of DNA, Yang will be able to measure the sequence and timing of transcription for the first time.
Ultimately, Yang's work could result in advances in disease research, drug design, turbulence, materials analysis, and even our grasp of basic biochemical reactions. Says Yang, "I hope that in doing these experiments, we will get the chance to know how nature makes these things happen, and take that understanding to improve our quality of life."