Factorially designed crystallization trials of the full-length P0 myelin membrane glycoprotein. I. Precipitation diagram

摘要

P0 glycoprotein is the abundant membrane protein of myelin of the peripheral nervous system. We report now the statistical design of the crystallization experiments; based on our belief that important information regarding supersaturation of protein or its solubility nature, as well as metastable state, nucleation or precipitation, are hidden in the trials in which no crystals grow. It is possible to work out this information when the whole set of experiments is designed in such a way as to allow statistical analyses. We selected seven factors, which we believe to be important for crystallization: protein concentration, pH of buffer, nature of precipitant, concentration of precipitant, nature of detergent, additives and temperature. The experimental matrix and detailed work sheet to make 148 solutions having random but balanced combination of these levels were calculated using the program DESIGN. A visual evaluation of crystallization drops was performed using light microscopy. We were able to plot the precipitation boundary diagram. Based on this diagram we have eliminated factors (and levels) that were driving the protein into precipitation. It is known that the precipitation boundary is related to the solubility curves for protein crystals, in the neighborhood of which nucleation and further crystallization is most likely to occur. These conditions are currently being refined to identify important factors (or its levels) that can be crucial in obtaining large and well diffracting crystals. Full-length PO protein has never been crystallized for structural determination.