Atomic-force-microscopy studies of phase separations in macromolecular systems

Abstract

Atomic force microscopy ͑AFM͒ has been used to visualize events arising from the formation of intervening metastable phases at the surfaces of macromolecular crystals growing from solution. Crystals investigated were of the proteins canavalin, thaumatin, lipase, xylanase, and catalase, crystals of transfer RNA, and crystals of satellite tobacco mosaic 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 produced defects of various kinds. The second aggregate form we infer to be liquid-protein droplets which were particularly evident in freshly mixed protein-precipitant solutions. Droplets, upon sedimentation, have two possible fates. In some cases they immediately restructured as crystalline multilayer stacks whose development was guided by and contiguous with the underlying lattice. These contributed to the ordered growth of the crystal by serving as sources of growth steps. In other cases, liquid-protein droplets formed distinct microcrystals, somehow discontinuous with the underlying lattice, and these were subsequently incorporated into the growing substrate crystal with the formation of defects. Scarring experiments with the AFM tip indicated that liquid-protein droplets with the potential to rapidly crystallize were a consequence of concentration instabilities near the crystal's surfaces. The AFM study suggests that phase separation and the appearance of aggregates having limited order is a common occurrence in supersaturated macromolecular solutions such as the protein-precipitant solutions used for crystallization.

Role of clusters in nonclassical nucleation and growth of protein crystals

“…35). Although the link between the presence of mesoscopic clusters in solution and the de novo formation of looped macrosteps has certainly been suggested (34,35), no experimental proof is available that demonstrates causality between both observations.…”

“…Two different models have been postulated to account for the quasi-instantaneous formation of multilayer stacks: the microcrystal-sedimentation scenario (28,33) and the cluster-assimilation scenario (34,35). In brief, the former model assumes coincidental sedimentation of freshly bulk-nucleated microcrystals in an at-random orientation onto the macrocrystal, followed by a rapid reorientation to align with the underlying lattice leading to a flawless merging of both phases (36).…”

Macromolecular crystal growth as revealed by atomic force microscopy

“…Such a phenomenon fails, however, to explain the perfect alignment of all of the stacks with the underlying lattice and with one another. A second explanation, for which there is now substantial and persuasive evidence, suggests that they arise from liquid protein phase droplets that exist in concentrated macromolecular solutions (Asherie et al, 1996;Kuznetsov et al, 1998;Lui et al, 1995). These liquid protein phase droplets are composed of many thousands of molecules exhibiting short-range order, mediated principally by nonspecific hydrophobic interactions and random arrangements of hydrogen bonds.…”

“…These form a crystal layer, inspire crystallinity in the molecules above them, and so forth, propagating a continuous series of growth layers, a multilayer stack. For some crystals for which the process was relatively slow, this phenomenon has been visually recorded (Kuznetsov et al, 1998).…”

Introduction to protein crystallization

“…Currently, the critical nuclear size has only been described for a few systems, and for several cases these were only investigated in terms of two-dimensional nuclei developing on the surfaces of already existent crystals (Malkin et al, 1996(Malkin et al, , 1997. Recently, a theory has emerged which attempts to explain the nucleation phenomenon in terms of statistical fluctuations in solution properties (Ten Wolde & Frenkel, 1997;Haas & Drenth, 1999;Piazza, 1999;Kuznetsov et al, 1998). This idea holds that a distinctive 'liquid protein phase' forms in concentrated protein solutions and that this 'phase' ultimately gives rise to critical nuclei with comprehensive order.…”