This proposal addresses the methodology employed for obtaining protein crystals for structural genomics or other purposes. Understanding the relationship between macromolecule structure and function is critical to an understanding of the molecular aspects of physiological functions essential to human health. This proposal is to test and develop the use of trace labeling of macromolecules undergoing crystallization screening for analyzing the results obtained. This proposal is to: 1) Demonstrate that the trace fluorescently labeled protein can be used to facilitate the crystal screening process, and determine a practical modification range for use in visual crystal detection and scoring of experimental results; 2) Develop facile protocols for making fluorescent derivatives that will readily fit within high-throughput purification and preparation procedures; 3) Determine the usable range for labeling macromolecules, from the minimal amount for detection to the maximum level, where X-ray data or crystallization is affected; 4) Test the utility of the labeled crystals for automated crystal alignment at synchrotron beamlines. The primary means of attaining these goals will be by comparative crystallization trials followed by X-ray analysis of the crystals obtained. Trace fluorescent labeled proteins will be provided to a synchrotron beamline (Argonne Nat. Lab.) for use in developing automated alignment methods. By covalently modifying a subpopulation of a protein solution with a fluorescent probe, the labeled population will add to a growing crystal as a microheterogeneous growth unit. The probe will concentrate in the crystal relative to the solution, and under fluorescent conditions they will show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity may also be used to distinguish previously obscure crystallization lead conditions. The fluorescent labeled crystals will also emit with sufficient intensity to aid in the automation of crystal mounting using relatively low cost optics, further increasing throughput at synchrotrons. While this proposal focuses on a visual approach, it is anticipated that success will subsequently lead to development of automated methods of analysis, more suitable for high throughput application. ? ?
| Pusey, Marc; Forsythe, Elizabeth; Achari, Aniruddha (2008) Fluorescence approaches to growing macromolecule crystals. Methods Mol Biol 426:377-85 |
| Forsythe, Elizabeth; Achari, Aniruddha; Pusey, Marc L (2006) Trace fluorescent labeling for high-throughput crystallography. Acta Crystallogr D Biol Crystallogr 62:339-46 |
