Determining the molecular-growth mechanisms of protein crystal faces by atomic force microscopy

Abstract

A high-resolution atomic force microscopy (AFM) study has shown that the molecular packing on the tetragonal lysozyme (110) face corresponds to only one of two possible packing arrangements, suggesting that growth layers on this face are of bimolecular height [Li et al. (1999). Acta Cryst. D55, 1023-1035]. Theoretical analyses of the packing also indicated that growth of this face should proceed by the addition of growth units of at least tetramer size, corresponding to the 43 helices in the crystal. In this study, an AFM linescan technique was used to measure the dimensions of individual growth units on protein crystal faces as they were being incorporated into the lattice. Images of individual growth events on the (110) face of tetragonal lysozyme crystals were observed, shown by jump discontinuities in the growth step in the linescan images. The growth-unit dimension in the scanned direction was obtained from these images. A large number of scans in two directions on the (110) face were performed and the distribution of lysozyme growth-unit sizes were obtained. A variety of unit sizes corresponding to 43 helices were shown to participate in the growth process, with the 43 tetramer being the minimum observed size. This technique represents a new application for AFM, allowing time-resolved studies of molecular processes to be carried out.

Direct Observation of Adsorption Sites of Protein Impurities and Their Effects on Step Advancement of Protein Crystals

“…We observed in situ the impurity effects of F-lysozyme, dimer, 18 kDa, and impurities included in Seikagaku lysozyme (98.5% purity) on the morphology of elementary 2D islands on {110} faces of tetragonal lysozyme crystals by LCM-DIM. As shown in Figure 2, 2D islands with elementary step height (5.6 nm) [46][47][48] could be clearly visualized by LCM-DIM. 11,33,36,37,44 The 2D islands formed in a 99.99% purity solution (Figure 2a) are lens-shaped with sharp tips.…”

Effect of an External Electric Field on the Kinetics of Dislocation-Free Growth of Tetragonal Hen Egg White Lysozyme Crystals

“…Here s = 7.84 × 10 −18 m 2 [69]. Substituting Equation (4) into Equation (3) gives the growth rate in the coordinates ln(R/σ 1/6 (1 − e −σ ) 2/3 ) versus 1/σT 2 on the (110) face as follows:…”

“…On the other hand, in the case of the (101) faces, an elementary step involves one molecule [66][67][68][69]; therefore, the rate of 2D nucleus formation J on the (101) face can be expressed according to the classical nucleation theory [62,70]:…”

“…where is the frequency of attachment of molecules to the critical 2D nucleus, Γ is the Zeldovich factor, Z is the steady-state admolecule surface concentration, s is the area in which one molecule occupies inside the critical 2D nucleus (s = 1.06 × 10 −17 m 2 ) [69], κ is the specific edge free energy, k B is the Boltzmann constant, and T is the absolute temperature.…”

Determination of crystal orientation from micrographs using a MATLAB program

“…Note that the crystal in this instance grew attached to the base of the crystallization plate, so that the top faces of the crystal are used to determine the proper alignment. For crystals of Crystallographic faces and directions of lysozyme (after Li et al, 1999).…”