蛋白晶体学lecture1.ppt

Four major steps in crystallization Obtain large amounts of pure protein samples Choose a protein buffer in which the protein is both soluble and stable Bring protein solution to supersaturation where spontaneous nucleation can take place Crystal growth now begins Still no crystals after thorough screening. Now what? New constructs Deletion mutants Complexes with substrates Protein complex with Fab fragments Homologous proteins Crystallization of membrane proteins The lipidic cubic phase method (Landau and Rosenbusch) Cocrystallization with Fab fragments Properties of protein crystals Soft, easy to crush Contain large solvent channels Relatively large organic and inorganic molecules can diffuse inside Anisotropic physical properties Birefrigence due to anisotropic refraction indices Ability to diffract X-ray due to regular spaced lattices * Lecture 1: Crystallization Methods and Protein Crystal Properties Solubility As a rule, protein solubility will usually increase as you add salt to your aqueous solution, then begin to decrease when the salt concentration gets high enough to compete with the protein for hydration (interaction with water molecules). Diagram from the website of Alan Clark, Victoria University of Wellington, New Zealand http://www2.vuw.ac.nz/staff/alan_clark/teaching/index.htm HbCO (carboxyhemoglobin) solubility as a function of ionic strength in the presence of several different types of salts Supersaturation Supersaturation can be achieved by adding more of a substance (to a solution) than can normally be dissolved. This is a thermodynamically unstable state, achieved most often in protein crystallography by vapor diffusion or other slow evaporation techniques. Zone 1 - Metastable zone. The solution may not nucleate for a long time but this zone will sustain growth. It is frequently necessary to add a seed crystal. Zone 2 - Nucleation zone. Protein crystals nucleate and grow. Zone 3 - Precipitation zone. Proteins d