Mapping electrostatic potential of a protein on its hydrophobic surface: implications for crystallization of Cytochrome P450scc

Calculation and combined visualization of electrostatic and hydrophobic properties of Cytochrome P450scc based on two very different homology models allowed to identify extensive hydrophobic patches with neutral electrostatic potential and mutations removing such patches and thus expecting to facilitate crystallization of Cytochrome P450scc, especially for the nanotemplate crystallization method. Implications are discussed for optimizing crystallization and other aspects of protein surface properties and protein recognition.     

A new method for the preparation of giant liposomes in high salt concentrations and growth of protein microcrystals in them

We have developed a new method for the preparation of giant liposomes in aqueous solution containing high salt concentrations (up to 2.0 M). Hydrophilic polymers attached to the surface of lipid membranes by including a small amount of poly(ethylene glycol)-grafted phospholipid in the membrane increase the repulsive force between the membranes, which makes it possible to form giant liposomes at high ionic strength. Using this method, we could grow micron-sized (10-50 μm) protein crystals in a giant liposome. These results demonstrate that this method is a promising tool for the preparation of ‘artificial cells’ under various conditions.     

Scanning probe acoustic microscopy of extruded starch materials: Direct visual evidence of starch crystal

Scanning probe acoustic microscopy (SPAM) has been successfully used to study inorganic and keratin biomaterials. However, few studies have attempted to apply SPAM to structural study of non-keratin organic materials such as starch based materials. This study investigated hardness and surface finish to establish sample preparation method suitable for SPAM imaging and acquired clear acoustic images of extruded starch materials. Acquired acoustic images directly exhibited certain structure of starch materials and provided visual evidence of starch material components and aggregates. In addition, through correlating acoustic images with X-ray diffraction data, crystal-structural information in nano-scale was obtained and acoustic image contrast showed a linear relationship with starch amylose content in extruded starch materials.     

Four-dimensional microED of conformational dynamics in protein microcrystals on the femto-to-microsecond timescales

As structural determination of protein complexes approaches atomic resolution, there is an increasing focus on conformational dynamics. Here we conceptualize the combination of two techniques which have become established in recent years: microcrystal electron diffraction and ultrafast electron microscopy. We show that the extremely low dose of pulsed photoemission still enables microED due to the strength of the electron bunching from diffraction of the protein crystals. Indeed, ultrafast electron diffraction experiments on protein crystals have already been demonstrated to be effective in measuring intermolecular forces in protein microcrystals. We discuss difficulties that may arise in the acquisition and processing of data and the overall feasibility of the experiment, paying specific attention to dose and signal-to-noise ratio. In doing so, we outline a detailed workflow that may be effective in minimizing the dose on the specimen. A series of model systems that would be good candidates for initial experiments is provided.