Spotting Nascent Protein Crystals

A microscopy technique that harnesses a special optical effect could detect smaller protein crystals than can any other optical method. The technique may reduce the time and expense required to screen crystallization conditions, a major bottleneck in protein structure determination by X-ray crystallography.

Traditional high-throughput crystal screens can find only protein crystals that are at least micrometer-sized. Some screens also have a significant background signal due to fluorophores used in detection. Garth J. Simpson and coworkers at Purdue University have shown that their microscopy technique, based on a nonlinear optical effect called second harmonic generation (SHG), effectively eliminates background noise in crystal screens (J. Am. Chem. Soc., DOI: 10.1021/ja805983b). They estimate that their technique can discern crystals with dimensions in the 100-nm range. This improvement in resolution would reduce the amount of protein needed per screen and allow earlier detection without the need for fluorophores.

SHG occurs when intense laser light interacts with certain ordered materials, including some crystals. In such cases, some photons from the laser combine to form new photons with twice the energy of the originals. The Purdue team outfitted a microscope with a laser and detected crystals of two different proteins via their telltale SHG signals. "The advantage of our technique is its selectivity," Simpson says. Randomly oriented protein aggregates and solvent molecules cannot give off that signal, which eliminates background noise, he explains. The technique won't work on highly symmetrical protein crystals because of the physical principles governing SHG, but such crystals make up less than 1% of crystals that have been characterized, he adds.

"Since the majority of protein crystal classes should be SHG active, there is the potential for widespread adoption of this idea to screen for crystals," says bioanalytical chemist Paul S. Cremer of Texas A&M University.

"The fact that this work is performed with a standard laser system will make it highly attractive to the scientific community," adds spectroscopist Franz M. Geiger of Northwestern University.