Towards protein-crystal centering using second-harmonic generation (SHG) microscopy

Abstract

The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using 2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 mm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.

The effects of solution additives and gas‐phase modifiers on the molecular environment and conformational space of common heme proteins

“…Mass spectra show a narrow distribution of charge states: +5 to +8 for cyt C and +6 to +9 for Mb under neutral conditions (pH 6.7). Note that for a solution pH change from 5.0 to 7.0 the average charge state in solution (determined using PROPKA3 with 2B4Z and 4 DC8 reported crystal structures for cyt C and Mb, respectively) varies from ~12 to ~8 for cyt C and ~9 to ~1 for Mb, indicating that a variety of charge states are accessible in solution between mildly acidic and neutral pH conditions. In most cases, minor differences in the charge state distribution during TIMS‐MS were observed for cyt C and Mb as a function of the starting solvent conditions.…”

Interactions of arene ruthenium metallaprisms with human proteins

“…Moreover, high-resolution crystal structures or NMR structural data are available, making them ideal candidates for such an investigation. [36][37][38][39][40] Albumin, known to transport numerous important biomolecules and drugs in the cytoplasm, 41 and transferrin, which controls the level of free iron in biological fluids, may react immediately with the metallaprisms, if they are administrated intravenously. Cytosolic proteins also possess important functions: cytochrome c is involved in apoptotic pathways, 42 while ubiquitin is relevant in posttranslational modifications together with the proteasome system, 43 and myoglobin is the primary oxygen-carrying pigment of muscle tissues.…”

Influence of pH on permeability of ceramic membranes and selectivity in ultrafiltration of model BSA and myoglobin solutions

“…The starting structure of MG was obtained from XRD based 4DC8 pdb file [13] and the BSA structure from 4F5S pdb file [14] deposited at RCSB Protein Data Bank.…”