Small chips, big promise microfluidic platforms for protein crystallization

Embedded in the oily interior of the membrane boundaries that surround cells and intracellular organelles, such as mitochondria and cell nuclei, is a class of proteins called “membrane proteins.” With surfaces that communicate with either side of the boundary, these proteins carry out critical processes that are essential for health, such as the transport of glucose and other nutrients between blood and tissues, the conversion of electrochemical and chemical energy in mitochondria, and the conversion between electrical and chemical signals in nerve cells.

Many diseases such as Alzheimer’s, diabetes, cystic fibrosis, and hypertension have been linked to the malfunction of these membrane proteins. Designing effective new pharmaceuticals that address such diseases requires the knowledge of the 3-D structure of these proteins.

The most successful method of obtaining 3-D structures of proteins is by X-ray crystallography. In this method, purified proteins are coaxed into forming crystals that are then subjected to intense X-ray beams to obtain the necessary data. However, membrane proteins have proven to be extremely difficult to purify, and generally yield very small amounts after purification.

Finding the optimal conditions that can coax membrane proteins to form crystals is such a huge barrier, that currently the structures of non-membrane proteins, also called ‘soluble proteins,’ outstrips those of membrane proteins by 150 to 1, even though genomes encode approximately two soluble proteins for every membrane protein. Even when membrane proteins do form crystals, they often get damaged when being removed from the crystallization solution in preparation for X-ray analysis.

The challenge: Devise a method to enable crystallization using the smallest possible amount of purified protein while enabling X-ray analysis without the need to remove the crystals from the crystallization solution--and keeping the whole method cost-effective for any biochemical laboratory.

The solution: Researchers led by Paul Kenis, the William H. and Janet G. Lycan Professor and head of the Department of Chemical and Biomolecular Engineering, have developed novel microfluidic platforms that have the potential to greatly enhance protein crystallization success. In the August issue of Lab on a Chip, the researchers demonstrated the use of a X-ray transparent microfluidic chip for the complete structure determination of a novel protein in their research article, “A Microfluidic Approach for Protein Structure Determination at Room Temperature via on-chip Anomalous Diffraction.”

These chips can be used for the initial search for crystal-forming conditions, followed by optimization, and finally the collection of X-ray data by placing the entire chip, with hundreds of protein crystals within, in the path of intense X-rays. The synchrotron facility for X-ray beams used in this study is located at Argonne National Laboratory near Chicago, and the instruments there enable the targeting of individual protein crystals within the chip for X-ray analysis.

“The work done in the current publication establishes the strength of our technology as a single platform to carry out the complete set of experiments from testing for crystallization conditions to collecting high-quality X-ray diffraction data,” said Ash Pawate, one of the authors of the study, “We are working on making our chips available to any researchers who are interested.”

Sudipto Guha, former graduate student in the Kenis lab and a co-author of the study, said with the new chip, structural biology laboratories will be able to conduct crystallization experiments at a lower cost.

“Our platform will bring high-throughput protein crystallization and on-chip structure determination capabilities to labs at a fraction of the cost of expensive robotic systems that are otherwise needed,” Guha said.

“The ability to enhance the available knowledge of challenging proteins, such as those responsible for diseases, disease transmission, or those which could be potential targets for pharmaceutical intervention have tremendous potential to enhance not only our biochemical understanding of the science behind the disease and treatment, but also to improve the quality of life for people around the world,” said Sarah Perry, a former graduate student and co-author.

The authors of the study include former graduate students Sarah Perry (now a postdoctoral researcher at the University of Chicago) and Sudipto Guha (currently at Intel in Portland, Oregon), research scientist Ash Pawate, undergraduate student Amrit Bhaskarla, graduate student Vinayak Agarwal (currently a postdoctoral researcher at the Scripps Institute of Oceanography in San Diego, California) and his advisor, Satish Nair (biochemistry), and Paul Kenis.