Real-time investigation of dynamic protein crystallization in living cells

Abstract

X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus μNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitro.

In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors

“…Most crystals regularly exceed the normal dimensions of Sf9 cells (20-25 μm), without affecting cell viability ( Fig. 1c, d, Supplementary Movie 1), as previously observed for TbCatB 31 and firefly luciferase 32 .…”

“…TbCatB and TbIMPDH do not share any similarities other than the source organism. However, within 3-6 days after baculovirus infection of the insect cells, the intracellular crystallization process consistently led to the formation of needleshaped crystals comparable to those observed for firefly luciferase and reoviral GFP-μNS proteins 32 a more general crystallization mechanism that can be exploited for other proteins as well. A prolific interplay between high local protein concentrations and the intrinsic crystallization tendency of the target proteins at specific cellular conditions has already been suggested to favor in cellulo crystal growth 7,10 .…”

Protein phase separation and determinants of in cell crystallization

“…They demonstrated that growth of in vivo crystals is a highly dynamic process and that these crystals were located either inside of peroxisomes or within the cytosol, respectively. So far, characterization of firefly luciferase using scanning electron microscopy (SEM) and GFP‐μNS using X‐ray powder diffraction studies have been reported . Tsutsui et al reported expression of an Xpa Coral protein in HEK293 cells, were crystals were encapsulated by autophagosome/lysosomal membranes (although some crystal‐like structures were also found in the nucleus).…”

“…Further, in cellulo crystallization has also been associated with several diseases like cataract, hemoglobin C diseases, formation of Charcot‐Leyden crystals (CLCs), Reinke's crystals or mitochondrial myopathies, and more recently in cellulo crystallization was observed as a result of heterologous overexpression of genes in cell lines of bacteria, insect cells, yeast, CHO (Chinese hamster ovary) or HEK (human embryonic kidney) cells . Mostly, these protein crystals were located in different organelles (mitochondria, peroxisomes, lysosomes, or endoplasmic reticulum), as shown in Table but sometimes also within the cytosol or even in the nucleus, as shown in Figure and Table .…”

“…Therefore, protein sorting into organelles with limited space seemed originally a prerequisite for in vivo crystallization. However, occurrence of in vivo crystals within the cytosol may also reflect preceding LLPS events. To reach the required protein supersaturation for nucleation and crystal growth, phase separation is supported by crowding agents in the cell, which promote attractive protein‐protein interactions and act similar to precipitants in in vitro experiments .…”