SBIR-STTR Award

Structural information of proteins have shown to be of critical importance in the discovery and development of pharmaceuticals. Membrane proteins such as ion channels and G-protein coupled receptors constitute a sizeable fraction of human genes and are targeted by approximately 60% of drugs that are currently on the market. Determinatinations of the 3-D structures of membrane proteins have lagged far behind the determination of structures of soluble proteins. A recently developed technique for membrane protein crystal growth involves the use of a crystallization matrix that consists of a lipidic cubic phase in which the membrane protein crystallizes in a microenvironment resembling native lipid bilayers. So far only colored membrane proteins have been crystallized using this method. This is partly due to the ease of detection of colored crystals in an otherwise colorless background. Though optically transparent and non-birefringent, the detection of non-colored crystals in these lipidic phases poses a challenge. The reason for this lies in the generally small size of crystals (sometimes less than 50 micrometers), in their low contrast and in optical obstructions caused by lipid phase transitions that may occur during the course of the crystallization experiment. The goal of this project is to develop a microscope-based imaging technology that alleviates this limitation. It is proposed to construct an automated, digitally enhanced cross-polarization microscope for the detection of colorless microcrystals. Ultimately this digital contrast enhancement technology will allow fast and efficient detection of non-colored membrane protein crystals in crystallization trials.

Thesaurus Terms:
biomedical equipment development, crystallization, digital imaging, light microscopy, optical polarization, protein quantitation /detection bioengineering /biomedical engineering, bioimaging /biomedical imaging

We propose to investigate, design and manufacture a sophisticated, pre-production protein crystal detection microscope, the Crystal Finder(tm) microscope workstation, incorporating extinction polarization and quantitative birefringence imaging for digitally enhanced display of crystal bulk contrast (referred to herein as Detect-X(tm) technology). Many protein crystallization experiments are of the trial-and-error type, where frequently only one in 1,000 experiments provides a crystal hit. Often these initial hits are difficult to detect because crystals may be small, colorless and disguised by amorphous precipitate material. Most commercial microscopes used to analyze protein crystallization setups provide crystal edge contrast only, which is not compatible with efficient screening of colorless membrane protein crystals grown in lipidic cubic phases. Thus, there is a critical need for more reliable and rapid methods fort the accurate characterization of crystalline materials. To provide a solution to this problem, research conducted during Phase I of this project demonstrated the successful implementation of the Detect-X(tm) technology in a proof-of-concept instrument. This technology, allows detecting crystals by virtue of their bulk properties (birefringence) rather than their refractive index difference with the background (i.e. crystal edges and facets). This technology results in a higher number of accurately detected protein crystals, with a concomitant decrease in false positive hits. Specific Aim I: A pre-production automated polarization microscope, the Crystal Finder(tm) microscopic workstation with Detect-X(tm) technology will be designed and built by Appalachian Electronic Instruments (Fairlea, WV). Specific Aim II: A user friendly instrument control interface and optimized Detect- X(tm) software will be designed and implemented by Emerald BioSystems (Bainbridge Island, WA). This software will be integrated into existing Crystal Miner(tm) crystallization tracking software and relational database. Specific Aim III: The instrument will be evaluated for performance in imaging of challenging protein crystals in a crystallization trial using digitally enhanced crystal detection. A side-by-side comparison with competing commercial protein crystal imaging instruments will be carried out. It is anticipated that the Crystal Finder(tm) microscope workstation will dramatically impact the successful identification of difficult-to-detect protein crystals in crystallization trials. The unique features and superior performance of the Crystal Finder(tm) microscopic workstation provides the basis of its commercial success. It is expected to set the standards for automated high-throughput image-enhanced protein crystallization experiment screening and will be the instrument of choice for both large consortiums and medium-size research laboratories. Protein structure is a cornerstone in biological science and medicine providing a molecular basis for disease and drug action. The technology advancement described in this proposal, and built into a protein crystal detection microscope workstation, is expected to make a significant contribution to the needs of the protein crystallography community and public health. It is expected to accelerate the pace by which protein structures are solved, and will be a critical mediator of advancement in medical science and drug discovery