Heterogeneous nucleation of three-dimensional protein nanocrystals

Abstract

Nucleation is the rate-limiting step in protein crystallization. Introducing heterogeneous substrates may in some cases lower the energy barrier for nucleation and thereby facilitate crystal growth. To date, the mechanism of heterogeneous protein nucleation remains poorly understood. In this study, the nucleating properties of fragments of human hair in crystallization experiments have been investigated. The four proteins that were tested, lysozyme, glucose isomerase, a polysaccharide-specific Fab fragment and potato serine protease inhibitor, nucleated preferentially on the hair surface. Macrocrystals and showers of tiny crystals of a few hundred nanometres thickness were obtained also under conditions that did not produce crystals in the absence of the nucleating agent. Cryo-electron diffraction showed that the nanocrystals diffracted to at least 4 A resolution. The mechanism of heterogeneous nucleation was studied using confocal fluorescent microscopy which demonstrated that the protein is concentrated on the nucleating surface. A substantial accumulation of protein was observed on the sharp edges of the hair's cuticles, explaining the strong nucleating activity of the surface.

Protein crystallization facilitated by molecularly imprinted polymers

“…13); however, this requires the availability of crystals of the given protein or at least some crystalline material to start with. In an ongoing search for alternative heterogeneous seeding materials, a variety of substances such as minerals (14), horse (15) and human (16) hair, thin films (17), charged surfaces (18,19), mesoporous materials (20)(21)(22), and other materials (23) have been used as nucleants with varied success. The problem with such materials is that they are random substances, which have helpful properties such as porosity, nanostructure, or electrostatic attractive potential, but no designed specificity for proteins.…”

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal

“…We could improve the SNR substantially with a more accurate and sensitive detector. Previously, we measured three-dimensional nanocrystals similar to the polymorph presented here using CCD detectors and image plates (Georgieva et al, 2007(Georgieva et al, , 2011. For protein crystals that had a similar diffracting volume to that reported here, we could never measure more than a few diffraction patterns of high-resolution data with a CCD detector or image plate before radiation damage became too severe.…”

The strong phase object approximation may allow extending crystallographic phases of dynamical electron diffraction patterns of 3D protein nano-crystals

“…When protein crystals fail to grow to a size sufficient for X-ray crystallography, electron diffraction may be a viable alternative (Georgieva et al, 2007). The reason why electron diffraction is a more attractive option for very small crystals than X-ray diffraction, is that for each elastically diffracted quantum, electrons on average deposit less energy in the sample than X-rays (Henderson, 1995).…”

“…For resolutions up to 2 A at electron energies between 100 keV to 300 keV (with relativistic electron wavelengths varying between l r % 0:035 A and l r % 0:015 A, respectively), the curvature of the Ewald sphere can be ignored. Resolutions up to 2 A can be achieved for 3D protein crystals (Georgieva et al, 2007). If we ignore the curvature of the Ewald sphere, the structure factors Fðh hÞ of the exit wave function wðx xÞ are defined by a Fourier transform 2 :…”