ATOMIC-FORCE-MICROSCOPY STUDIES OF PHASE SEPARATIONS IN MACROMOLECULAR SYSTEMS

Atomic force microscopy (AFM) has been used to Visualize events arisin g from the formation of intervening metastable phases at the surfaces of macromolecular crystals growing from solution. Crystals investigate d were of the proteins canavalin, thaumatin, lipase, xylanase, and cat alase, crystals of transfer RNA, and crystals of satellite tobacco mos aic virus. Two types of aggregates were observed. The first were small , linear and branched aggregates, perhaps fractile in structure. These were incorporated into growing crystals as impurities, and they produ ced defects of various kinds. The second aggregate form we infer to be liquid-protein droplets which were particularly evident in freshly mi xed protein-precipitant solutions. Droplets, upon sedimentation, have two possible fates. In some cases they immediately restructured as cry stalline multilayer stacks whose development was guided by and contigu ous with the underlying lattice. These contributed to the ordered grow th of the crystal by serving as sources of growth steps. In other case s, liquid-protein droplets formed distinct microcrystals, somehow disc ontinuous with the underlying lattice, and these were subsequently inc orporated into the growing substrate crystal with the formation of def ects. Scarring experiments with the AFM tip indicated that liquid-prot ein droplets with the potential to rapidly crystallize were a conseque nce of concentration instabilities near the crystal's surfaces. The AF M study suggests that phase separation and the appearance of aggregate s having limited order is a common occurrence in supersaturated macrom olecular solutions such as the protein-precipitant solutions used for crystallization.