Engineering of protein crystals for use as solid biomaterials

Protein crystals have attracted a great deal of attention as solid biomaterials because they have porous structures created by regular assemblies of proteins. The lattice structures of protein crystals are...

Why Do Protein Crystals Grown With the Aid of Porous Materials Show Better Diffraction Quality?

Well-diffracting protein crystals are indispensable for X-ray diffraction analysis, which is still the most powerful method for structure-function studies of biomolecules. A promising approach to growing such crystals is by using porous nucleation-inducing materials. However, while protein crystal nucleation in pores has been thoroughly considered, little attention has been paid to the subsequent growth of the crystals. Although the nucleation stage is decisive, it is the subsequent growth of the crystals outside the pore that determines their diffraction quality. The molecular-scale mechanism of growth of protein crystals in and outside pores is here considered theoretically. Due to the metastable conditions, the crystals that emerge from the pores grow slowly, which is a prerequisite for better diffraction. This expectation has been corroborated by experiments carried out with several types of porous material, such as Bioglass (“Naomi’s Nucleant”), Buckypaper, porous gold and porous silicon. Protein crystals grown with the aid of Bioglass and Buckypaper yielded significantly better diffraction quality compared with crystals grown conventionally. In all cases, visually superior crystals are usually obtained. We furthermore conclude that heterogeneous nucleation of a crystal outside the pore is an exceptional case. Rather, the protein crystals nucleating inside the pores continue growing outside them.

Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization Conditions

Neutron diffraction experiments are informative for determining the locations of hydrogen atoms in protein molecules; however, much larger crystals are needed than those required for X-ray diffraction. Thus, additional techniques are required to grow larger crystals. Here, a unique crystallization device and strategy for growing large protein crystals are introduced. The device uses two micropumps to control crystal growth by altering the precipitant concentration and regulating the pinpoint injection of dry air flow to the crystallization cell. Furthermore, the crystal growth can be observed in real time. Preliminary microbatch crystallization experiments at various concentration ranges of polyethylene glycol (PEG) 4000 and sodium chloride were first performed to elucidate optimized crystallization conditions. Based on these results, a device to precisely control the sodium chloride and PEG concentrations and the supply of dry air to the crystallization cell was used, and 1.8 mm lysozyme and 1.5 mm alpha-amylase crystals with good reproducibility were obtained. X-ray data sets of both crystals were collected at room temperature at BL2S1 of the Aichi Synchrotron Radiation Center and confirmed that these crystals were of high quality. Therefore, this crystallization device and strategy were effective for growing large, high-quality protein crystals.

Damaged goods? Evaluating the impact of X-ray damage on conformational heterogeneity in room temperature and cryo-cooled protein crystals

X-ray crystallography is a cornerstone of biochemistry. Traditional freezing of protein crystals to cryo-temperatures mitigates X-ray damage and facilitates crystal handling but provides an incomplete window into the ensemble of conformations at the heart of protein function and energetics. Room temperature (RT) X-ray crystallography provides more extensive ensemble information, and recent developments allow conformational heterogeneity, the experimental manifestation of ensembles, to be extracted from single crystal data. However, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical question, we obtained increasingly X-ray-damaged high-resolution datasets (1.02–1.52 Å) from single thaumatin, proteinase K, and lysozyme crystals. Heterogeneity analyses indicated a modest increase in conformational disorder with X-ray damage. Nevertheless, these effects do not alter overall conclusions and can be minimized by limiting the extent of X-ray damage or eliminated by extrapolation to obtain heterogeneity information free from X-ray damage effects. To compare these effects to damage at cryo temperature and to learn more about damage and heterogeneity in cryo-cooled crystals, we carried out an analogous analysis of increasingly damaged proteinase K cryo datasets (0.9–1.16 Å). We found X-ray damage-associated heterogeneity changes that were not observed at RT. This observation and the scarcity of reported X-ray doses and damage extent render it difficult to distinguish real from artifactual conformations, including those occurring as a function of temperature. The ability to aquire reliable heterogeneity information from single crystals at RT provides strong motivation for further development and routine implementation of RT X-ray crystallography to obtain conformational ensemble information.