X-ray crystallography generate selegant, yet static, portraits for biologists' ever-growing gallery of protein structures. To reveal protein motion, bio-physicists have developed site-directed spin labeling (SDSL), which uses electron paramagnetic resonance (EPR) spectroscopy to probe protein dynamics. The technique, performed using an EPR spectrometer, is complementary to nuclear magnetic resonance (NMR) but also offers some distinct advantages, including higher sensitivity, applicability to larger proteins, and the potential for real-time measurements.
With SDSL, scientists can observe proteins in motion, "like watching a movie," says Wayne Hubbell of the University of California, Los Angeles, who originated the method in the 1980s. Since that time, SDSL has become "mature to the point where it is usable as routine standard technology," he says. Sunil Saxena, who explores new SDSL methodologies at the University of Pittsburgh, concurs. "It's backed by very solid theory," he says, which means that the software and instrumentation can now be "packaged...
EPR technology takes advantage of the unpaired electrons found in certain naturally occurring compounds, such as transition metal ions and free radicals. These electrons absorb radiation in the microwave range in response to an applied magnetic field. Unpaired electrons are rarely found in naturally occurring proteins, but with genetic engineering, paramagnetic compounds known as "spin labels" can be attached at specific amino acid positions.
The most commonly used spin-labeling reagent, (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate, contains a paramagnetic nitroxide side chain attached to a methanethiosul-fonate moiety, which reacts specifically with cysteine residues. The cysteine side chain is small, so cysteines can be substituted at nearly any position without loss of protein function. The unpaired electrons in these labels act as "tiny, tiny magnets" that have "lots of interactions with each other" and with other parts of the protein, says Saxena. When proteins move, these interactions change, and the changes are reflected in the EPR spectrum.
The technique is conceptually similar to NMR, except that in NMR, the tiny magnets are the nuclei of atoms such as carbon and nitrogen that lie in the protein backbone and amino acid side chains. Because there are so many of these, NMR spectra and their interpretation are much more complicated, and only relatively small proteins can be investigated. SDSL can be applied to proteins of any size, says Hubbell. Also, NMR signals are much weaker, necessitating hours of measurements, which eliminates the possibility of real-time observation of protein movements, he says.
SDSL complements fluorescence spectroscopy techniques, in which fluorescent tags are attached to proteins. Such methods are more sensitive than SDSL (down to the single-molecule level) and use multiple wavelengths, says Saxena. But fluorescent tags are much larger than spin labels, he notes, and thus more likely to interfere with the protein's native structure and movement.
At present, Hubbell's protein movie is more figurative than literal. But he sees a future in which it may be possible to build "a microscope that can see a single electron spin." Such an instrument is one goal of the ongoing MOSAIC (Molecular Observation, Spectroscopy, and Imaging using Cantilevers) project currently underway with funding from the Defense Advanced Projects Research Agency. If this project succeeds, SDSL will provide a new level of resolution at which researchers can observe proteins going about their daily business.
- Megan M. Stephan
The structure of the nitroxide side chain (spin label) most commonly used in the study of protein structure and dynamics. The electron density on the side chain (cyan net) is derived from the X-ray diffraction of crystals of T4 lysozyme with the spin label at position 44. The protein helices are represented as ribbons.