Attachment and Detachment Processes of Individual Lysozyme Molecules on a Surface of a Monoclinic Lysozyme Crystal Studied by Fluorescent Single-Molecule Visualization

On a {10 (1) over bar} face of a monoclinic crystal of hen egg-white lysozyme (HEWL), we visualized the attachment and detachment processes of individual fluorescent-labeled HEWL (F-HEWL) molecules by a fluorescent single-molecule visualization technique. We measured the changes in number density of F-HEWL molecules, whose positions were not changed for longer than a certain residence time, as a function of an adsorption time. We first confirmed that under an equilibrium condition, there was an induction period (similar to 120 min) of the attachment/detachment processes, during which period the number density remained constant. After the induction period, the number density increased linearly with the adsorption time, as it was recently found on a tetragonal HEWL crystal [Dai, G. L. Cryst. Growth Des 2011, 11 (1), 88-92]. In addition, we performed similar measurements under a supersaturated condition. Then we found that supersaturation significantly enhanced the attachment process after the induction time. The attachment/detachment processes finally reached a steady state, in which the attachment rate was higher than the detachment one. Moreover, we also found that in a rare case, an F-HEWL molecule adsorbed on a step laterally moved following the advancement of a growing step

Gradual Immobilization Processes of Molecules during Transitions from Solute to Solid States

The adsorption/desorption behavior of mobile solute molecules at a solution-crystal interface has been explored using crystals of model protein hen egg-white lysozyme (HEWL) and fluorescent-labeled HEWL (F-HEWL) molecules. We have tracked the transient processes occurring during adsorption/desorption of identical F-HEWL molecules on a tetragonal HEWL crystal surface by single-molecule visualization using a total internal reflection fluorescent microscope and pulsed discontinuous laser illumination. We found an induction period (similar to 70 min) after which the number density of F-HEWL molecules adsorbed mainly on steps increased linearly with the adsorption time. We show direct evidence that the residence time of molecules on the crystal surface gradually increases during the transition process from a solute species to the crystal after successive multistep processes

In situ observation of elementary growth processes of protein crystals by advanced optical microscopy

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined