To obtain comprehensive understanding of the effects of a solution flow, we directly measured the velocities of individual elementary steps under a forced solution flow by laser confocal microscopy combined with differential interference contrast microscopy. We also investigated the defect density of four kinds of model protein crystals grown with/without forced solution flow, by slightly etching the crystal surfaces and observing etch pits. It was clarified that the growth of spiral steps is less affected by impurities, and at appropriate solution flow velocity, the microdefects could be reduced by the flow. It is one reason the flow improves crystal quality. On contrary the dislocation was increased by the flow in three cases of model proteins, which indicate that a dislocation may not be a serious defect for the crystal quality. Furthermore, it is possible that the growth mechanism shift from 2D nucleation growth mechanism to spiral growth mechanisms as a result of the increased defects induced by the solution flow could indirectly explain why forced solution flow improves crystal quality. In this paper, we introduce our recent studies about protein crystal growth under the forced solution flow.
We observed in-situ individual fluorescence-labeled protein molecules, to reveal the dynamic behavior of protein molecules during diffusion, adsorption and desorption at an interface between a protein solution and a protein crystal. We found that protein molecules diffused along a crystal surface 4-5 orders of magnitude slower than in a bulk solution, indicating that the molecules strongly interacted with molecules that were aligned at the crystal surface. This result denotes that slow two-dimensional diffusion inside a range of interactions from the crystal surface is a general picture of surface diffusion at an interface between an aqueous solution and a hydrophilic crystal surface. We also found the existence of an induction period (〜70 min) after which the number density of protein molecules adsorbed on steps increased linearly with the adsorption time. We show direct evidences that the residence time of molecules on a crystal surface gradually increases during the transition process from a solute species to the crystal after successive multistep processes.
Photochemically induced protein crystallization by protein's multiphoton excitation based on enhanced field of localized surface plasmon resonance (LSPR) of gold nanostructures was investigated. As strong photons-molecules coupling fields, gold nanostructures composed of nanoblocks ware used. Crystallization probability depends on the excitation photon fluence, which indicates 3 photons absorption process occurred. We developed crystallization plates equipped with strong photon-molecule coupling field made of gold thin films that enhances crystallization frequency.
Control of the nucleation process of hen egg white lysozyme was demonstrated under application of an external alternating current (AC) electric field. This is attributed to the electrostatic energy added to the chemical potentials of the liquid and solid phases. The nucleation rate can be controlled by focusing on the difference in the permittivities between the liquid and solid phases, while the population of nucleated polymorphs can be regulated by focusing on the difference in the permittivities between two polymorphs. That is, the control of both an increase and decrease in the nucleation rate or the population of nucleated polymorphs was achieved by regulating the magnitude of those permittivities of each phase with various imposed frequencies, by utilizing a large dielectric relaxation of protein crystals.
Pressure is an attractive thermodynamic parameter. In this review, I present the great potentialities of high pressure for the promotion of studies on the enhancement of protein crystallization processes and correlation between functions and 3D structures of protein molecules. As a tool for enhancing the crystal growth, three of eight proteins show the decrease in its solubility under high pressure. Acceleration of growth and nucleation kinetics of glucose isomerase crystals occurred under high pressure. Step velocities under high pressure provided us direct information on activation volumes during incorporation processes of protein molecules at a kink site of crystals. Such activation volumes were negative in the case of glucose isomerase crystals. Precise discussion on the activation volume will be useful for understanding dehydration mechanisms during the incorporation processes. Usefulness of our standalone-type Be vessel for high-pressure protein crystallography was confirmed. With the vessel, precise high-pressure 3D structure analysis of protein crystals which are also grown under high pressure will be achieved.
Protein crystal growth reflects intermolecular interactions under hydration effects. Experimental and theoretical approaches to the stable face, polymorphs, and humidity-induced phase transitions are introduced. The first example is cubic insulin. We determined the stable crystal form and carried out in situ observation on surfaces by an optical microscope. In order to understand the stable form, intermolecular interactions were evaluated by the macrobond and the electrostatic energy transfer (EET) analyses. Although there are some difficulties in treatment of hydration effects, the calculated surface energy explains the stable form. The second example is polymorphs of ribonuclease A. Relationship between intermolecular interactions and crystallization conditions was discussed on the basis of polarity of contact surfaces. The last example is thaumatin, where decrement of water content induces crystal structural transformation. The decrease in water leads to a rise in salt concentration in the crystal, and, as a result, molecules might move to increase nonpolar contact area.
Mechanical properties such as elastic and plastic characteristics of protein crystals are important for the understanding of the intermolecular interaction and growth mechanism. However, there are very few studies on the mechanical properties of protein crystals because of the difficulty of the crystallization and handling. In this review, we discuss the measurements of elastic constants in protein crystals by ultrasonic pulse-echo method. All elastic constants in cross-linked hen egg-white lysozyme crystals with tetragonal structure at 98% relative humidity are determined to be C_<11>=C_<22>=5.50GPa, C_<12>=4.33GPa, C_<13>=C_<23>=3.94GPa, C_<33>=5.22GPa, C_<44>=C_<55>=0.68GPa, and C_<66>=0.84GPa, respectively. The elastic constants, especially shear components, of protein crystals are much smaller than those of organic molecular crystals with van der Waals bonding. The small elastic constants of protein crystals can be attributed to the intracrystalline water, especially mobile water, in the crystals. The control of the mobile water in protein crystals can lead to the growth of high quality crystals and the improvement of the crystal properties.