Proteins are the stars of the drama called life, but for years biologists only knew them through their publicity stills.
Researchers had developed techniques that could reveal the finest features of how a protein was put together, detailing the exact position of each atom in space. But they couldn't use that information to put those atoms in motion.
Biophysicist Ludwig Brand wanted to see the stars in action, to find a way to record proteins as they danced and flexed and tumbled. To detect and quantify these split-second movements, Brand, who is a professor of biology in the Krieger School of Arts and Sciences, helped develop and apply a technique known as fluorescent spectroscopy. The approach has advanced the investigation of topics as diverse as the immune system, programmed cell death and carbohydrate metabolism. His contributions were recently acknowledged by the Biophysics Society, which selected him as a Society Fellow.
"He's the absolute leader in this field internationally, a wonderful collaborator and friend," says Saul Roseman, Hopkins biology professor and frequent collaborator with Brand. Roseman compares the difference between more commonly used methods of analyzing protein structure and fluorescent spectroscopy to the difference between having a single image of a running horse and having multiple sequential images of the running horse.
"X-ray crystallography and nuclear magnetic resonance studies of protein structures are excellent places to start, but they don't really tell you that much about the dynamics of a protein, and fluorescent spectroscopy does," says Roseman, who enlisted Brand's help in his study of the compounds bacteria use to take in glucose, an important sugar. Their collaboration identified an interaction that activates a key enzyme.
Brand's techniques take advantage of a natural phenomenon known as fluorescence. He compares fluorescence to the phenomenon that makes watches and toys glow in the dark, phosphorescence. Shine a light on a material, switch it off, and the material will glow in a different color light. With proteins, though, the glow lasts only billionths of a second, not seconds or minutes.
Brand augments naturally occurring fluorescence in proteins with special probe molecules that are placed with the proteins under study, and can help signal changes in the shape of a protein.
He exposes the proteins to bursts of laser light that last only picoseconds, or trillionths of a second, and records and analyzes the energy the proteins emit in response, which is gone almost as quickly.
"If you looked at a protein when you turned the laser off, the fluorescence would seem to disappear immediately. But it isn't immediate. It's something that you can measure, and you can measure the shape of the curve as the intensity decreases," Brand says.
As proteins shed energy imparted by the laser, their physical and chemical properties, as well as their immediate surroundings, affect the way this energy is given off as light. Individual molecules can also affect fluorescence by transferring energy to each other, a process dependent on the distance between the molecules. Brand also can use fluorescence to measure the tumbling of proteins on short time scales.
On their own and with research partners in biology, biophysics, chemistry, medicine and engineering, Brand and the members of his lab have applied fluorescent spectroscopy to many different problems.
In a recent study related to the degenerative nerve disease multiple sclerosis, doctoral student Andrew Russo, now graduated, investigated the properties of myelin basic protein, an insulating protein found on the surface of nerve cells. In some MS patients, myelin becomes detached from nerve cells.
Russo and other researchers in Brand's lab built an artificial membrane to simulate a nerve cell membrane, and used fluorescent spectroscopy to study how the membrane interacted with myelin basic protein.
"We've been trying to approach this from a fundamental point of view, to understand what holds the protein onto the membrane," Brand says.
Late last year, Vikas Nanda defended his doctoral thesis on work completed in the Brand lab. Nanda set out to use fluorescence to study homeodomains, regions in proteins that bind to DNA to control gene activity. He soon confronted a surprise: Despite having a component frequently linked to fluorescence, the homeodomains he studied didn't fluoresce at all.
When Nanda investigated further, he found an unusual chemical bond that was dissipating the energy supplied by the laser, eliminating the need to shed the energy through fluorescence. To follow up, Nanda used protein structure databases to look for proteins with similar features. He found one in interleukin 2 (IL-2), a promoter of cell growth important to the immune system. Brand and Nanda are hopeful that the discovery will lead to new revelations about how IL-2 activates the immune response.
"The reason this lab is a really good place to do this kind of work is that Dr. Brand has not only had a lot of experience in biology in terms of working with proteins and all the biochemistry going on in the lab, but he also has a very physical way of looking at things," Nanda says.
With Hopkins research scientists Dmitri Toptygin and Norman Meadow, Brand is using fluorescence to study a protein involved in the transport of glucose into bacterial cells. What they learn may help infectious disease specialists working to develop new methods to control bacterial infections.
Some of the most important questions about the protein can be answered only by observing its dynamics over the course of billionths of a second, and Brand is quick to acknowledge his indebtedness to the rapid advance of high-speed light detectors. Many of the detectors were originally developed not for use in a biology lab but instead for particle detectors and other physics research facilities.
"Years ago, Leon Madansky, who was chairman of Physics, stopped here in our lab, and he looked at our equipment," Brand recalls, smiling as he remembers the visit from an old friend. His voice rises in mock astonishment as he remembers Madansky's reaction: "He said, 'I recognize this. We use this in high-energy physics. What are you guys doing with this?'
"Hopkins is a unique place to do this research, because it includes an outstanding medical school with extensive protein research and a great school of engineering, where the faculty are interested in fast lasers and detectors, and sometimes you find connections that aren't so obvious," Brand says.
He leaves the last part unspoken: Those unexpected connections sometimes pay off in unique and rewarding ways.