Selection of Protein Crystal Forms Facilitated by Polymer-Induced Heteronucleation

Abstract

Crystallization of biological macromolecules as high quality single crystals is critical for determining their structure and facilitates the rational design of drugs. Because macromolecules often crystallize in multiple phases that have unique diffraction properties, the selective production of phases is desirable. Furthermore, determining multiple structures allows for a greater understanding of the relationship between crystal packing and conformation. With the aim of exploiting the polymer-induced heteronucleation approach to selectively nucleate multiple macromolecule crystal forms, hen egg white lysozyme (HEWL) was chosen as a model. Selective phase production was achieved under conditions that, in the absence of added heteronuclei, result in crystallization of a single crystal form. Moreover, nucleation rate, which in turn affects the size and quality of HEWL crystals, was controlled by various polymer surfaces. Thus, the polymer-induced heteronucleation approach provides an additional diversity element which can be easily implemented to complement standard crystal growth techniques for the selective production of high quality protein crystals.

Figure 1

Photomicrographs of a) tetragonal, b) orthorhombic, c) monoclinic, and d) triclinic HEWL grown by their respective literature methods.

Figure 2

Photomicrographs showing the change in crystal size of tetragonal HEWL grown from the tetragonal-inducing condition in the presence of a) AA/MMA/DVB terpolymer, b) 2VP/MAN/DVB terpolymer, and c) DMAEMA/MAN/DVB terpolymer.

Figure 3

Photomicrographs showing a) the concomitant formation of monoclinic, tetragonal, and needle forms on glass, b) monoclinic crystals growing from EEMA/MMA/DVB terpolymer, c) tetragonal crystals growing from t-BuMA/TFPMA/DVB terpolymer, and d) needle crystals growing from HEMA/MMA/DVB terpolymer all crystallized using the monoclinic-inducing condition.

Figure 4

Raman spectra of five crystal forms of HEWL. Tetragonal, orthorhombic, monoclinic, and triclinic HEWL crystals utilized for analysis were grown by their respective literature procedures and needle-shaped crystals were grown from the monoclinic-inducing condition in the presence of polymers. Note the peak near 1050 cm^-1 for monoclinic, triclinic, and needles which indicates the presence of nitrate.

Figure 5

PXRD patterns of five crystal forms of HEWL.