Self-Assembly of Proteins at Interfaces and Two-Dimensional Protein Crystallization
EMSL Project ID
1644
Abstract
Research is on the role of specific protein-protein interactions on protein crystallization. We use a well characterized, two-dimensional (2D) crystal model system where the protein streptavidin forms 2D crystals beneath biotinylated lipid monolayers at an air/water interface. By making site-directed mutations to streptavidin, we can alter the protein-protein interactions and determine the effect on crystallization. Study is of interest to biophysics/ biochemistry,biosensors and biomaterials, may aid protein crystallographers in design of more efficient crystallization exper. We have computer models of various crystal contacts between proteins and identified several specific protein-protein interactions. Using these, we've designed several site-directed mutations that should affect crystal morphology and space group. We are generating the genes for our mutants, and we'll express and purify the mutant proteinsl using mass spec to characterize them. We'll grow 2D crystals of the mutant proteins and compare them to wild-type proteins. We observe/characterize crystal morphology and density via quantitative Brewster angle microscopy (BAM), allowing us to follow the crystal growth in real time and in the absence of any dyes or stains; however our resolution is limited. Since we cannot determine crystallographic information with BAM, we will transfer the crystals to carbon coated TEM gri! ds. By staining the crystals with uranyl acetate and imaging with TEM, we'll be able to determine the unit cell parameters of the crystals. We've used these techniques successfully (Edwards et al, Langmuir 14:4683-4687). We're setting up a new lab to perform our 2D protein crystallization research, and no longer have access to a transmission electron microscope. We want your microscopy facilities to image our crystals. Sample prep/ disposal at Whitman Coll.' multiple samples brought in batches.
Project Details
Project type
Exploratory Research
Start Date
1999-08-04
End Date
2000-06-30
Status
Closed
Released Data Link
Team
Principal Investigator