Stability and Radiation Damage of Protein Crystals as Studied by Means of Molecular Dynamics and Monte Carlo Simulation

Molecular Dynamics (MD) and Monte Carlo (MC) simulations of crystals can help in interpretation of experimental X-ray crystallography data. Particularly, they can be useful for understanding how various crystallization techniques affect protein conformational plasticity within the crystal lattice and the stability of biomolecular crystals. The latter has become especially important since the modern and extremely intense X-ray radiation sources (such as free electron lasers, FELs) appeared recently. In the present study we were able to show by means of computer simulations that the lysozyme crystals obtained using the Langmuir-Blodgett technique have an advantage over the classical ones (\u201cHanging Drop\u201d) in terms of their thermal stability as well as their stability against the radiation damage. We also demonstrate an important role of crystal water dynamics for stability of protein crystals

Protein nanocrystallography : Growth mechanism and atomic structure of crystals induced by nanotemplates

Protein nanocrystallography, a new technology for crystal growth based on protein nanotemplates, has recently been shown to produce diffracting, stable and radiation-resistant lysozyme crystals. This article, by computing these lysozyme crystals' atomic structures, obtained by the diffraction patterns of microfocused synchrotron radiation, provides a possible mechanism for this increased stability, namely a significant decrease in water content accompanied by a minor but significant \u3b1-helix increase. These data are shown to be compatible with the circular dichroism and two-dimensional Fourier transform spectra of high-resolution H NMR of proteins dissolved from the same nanotemplate-based crystal versus those from a classical crystal. Finally, evidence for protein direct transfer from the nanotemplate to the drop and the participation of the template proteins in crystal nucleation and growth is provided by high-resolution NMR spectrometry and mass spectrometry. Furthermore, the lysozyme nanotemplate appears stable up to 523 K, as confirmed by a thermal denaturation study using spectropolarimetry. The overall data suggest that heat-proof lysozyme presence in the crystal provides a possible explanation of the crystal's resistance to synchrotron radiation

Improved Success of Sparse Matrix Protein Crystallization Screening with Heterogeneous Nucleating Agents

Crystallization is a major bottleneck in the process of macromolecular structure determination by X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth generally occurs spontaneously in a supersaturated solution as a result of homogenous nucleation. However, in a typical sparse matrix screening experiment, precipitant and protein concentration are not sampled extensively, and supersaturation conditions suitable for nucleation are often missed

From art to science in protein crystallization by means of thin film technology \u2013 cytochrome P450scc crystals

A new method of protein nucleation and crystallization based on Langmuir-Blodgett (LB) technology was utilized for the template stimulation of crystal growth of so far non-crystallized proteins. Microcrystals (60 - 120 micron) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method under all crystallization conditions including those failing to lead to crystal formation in solution. The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in earlier study with a model system (lysozyme) as an unexpected acceleration and enhancement in the crystal growth. The presentation is focused on bovine cytochrome P450scc crystallization and subsequent crystals characterization by Atomic Force Microscopy under appropriate chamber. The results are discussed in terms of a possible transition in protein crystallization from art to science by means of thin film nanotechnology

Langmuir-Blodgett nanotemplates for protein crystallography

T he new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the LangmuirBlodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the airwater interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISASAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISASAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 110 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods

Atomic Force Microscopy Of Protein Films And Crystals

A new AFM instrument dedicated to protein crystal imaging in solution is here successfully introduced, as proven by its testing on both the crystals and the Langmuir-Blodgett films of two proteins having quite different molecular weight, namely chicken egg white lysozyme and bovine serum albumin. The AFM consists of a custom measuring head with a flexible SPM controller in house produced which effectively drives the head for contact, non-contact and spectroscopy modes. By providing the user with full control and monitoring of signals at the front panel we have achieved an high degree of customisation optimal for the protein crystal measurements here described. This approach allows to study the crystal periodicity and morphology in their mother liquid. In this way, protein crystal structure is not destroyed, as in the case of drying, and one can thereby investigate its periodic structure in its \u201cnatural aqueous\u201d environment. Comfortingly it appears to distinguish the protein crystals from the salt crystals, which under the optical microscope are frequently quite similar and often their difference is revealed only during X-ray analysis. Finally the AFM estimates of the given single proteins packing, order and morphology appear quite similar in the LB thin film and in the crystals, thereby allowing routine crystal measurements at high resolution reproducing previous report on membrane protein bovine cytochrome P450scc similarly studied in LB films and crystals. Our intention for the future is to attempt the exploitation of the use of optical tweezers for both AFM and microGISAXS studies of protein crystal growth, considering the recent proven ability to utilize the birefringence of lysozyme crystals to control their orientation and to monitor their growth in optical tweezers