Protein crystallization in living cells

Abstract

Protein crystallization in living cells has been observed surprisingly often as a native assembly process during the past decades, and emerging evidence indicates that this phenomenon is also accessible for recombinant proteins. But only recently the advent of high-brilliance synchrotron sources, X-ray free-electron lasers, and improved serial data collection strategies has allowed the use of these micrometer-sized crystals for structural biology. Thus, in cellulo crystallization could offer exciting new possibilities for proteins that do not crystallize applying conventional approaches. In this review, we comprehensively summarize the current knowledge of intracellular protein crystallization. This includes an overview of the cellular functions, the physical properties, and, if known, the mode of regulation of native in cellulo crystal formation, complemented with a discussion of the reported crystallization events of recombinant proteins and the current method developments to successfully collect X-ray diffraction data from in cellulo crystals. Although the intracellular protein self-assembly mechanisms are still poorly understood, regulatory differences between native in cellulo crystallization linked to a specific function and accidently crystallizing proteins, either disease associated or recombinantly introduced, become evident. These insights are important to systematically exploit living cells as protein crystallization chambers in the future.

A Bacterial Surface Layer Protein Exploits Multi-step Crystallization for Rapid Self-assembly

“…The structural determinants of macromolecular phase transitions are important to govern a wide variety of biological processes and disease states (1, 2). Microbial SLPs are highly expressed and ubiquitous proteins that self-assemble on the outside of bacteria and archaea to form crystalline protein coats (3–5).…”

Protein phase separation and determinants of in cell crystallization

“…Interestingly, most in vivo crystals have a needle‐like shape, beside a few cubic‐rhomboid, hexagonal and bipyramidal morphologies reported till now and they are mainly located within the cytosol or inside membrane surrounded organelles like the endoplasmic reticulum, mitochondria, lysosomes and peroxisomes as shown in Figure . Details about in vivo crystallization and crystals observed in cellulo are also reported by Doye and Poon, Duszenko et al, Schönherr et al and Hasegawa …”

“…For more than a century it has been observed that protein crystallization occurs within living cells . In Table we summarize selected examples of in vivo protein crystallization together with their intracellular location inside different organism or cell systems.…”

“…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 .…”

“…In context of liquid‐liquid phase separation (LLPS) of biomolecules into liquid condensates in cells occasionally protein crystals were observed . The observation of crystals in living cells, reported in the literature as in cellulo or in vivo crystals is known for decades and was repeatedly described as a natural phenomenon . Examples are crystals of storage proteins in seeds, insulin crystals within secretory granules, solid state catalysts, biocrystallization of the DNA‐binding protein (Dps) and DNA as a response to cellular damage and stress, or β‐hematin crystals produced as a detoxification strategy by malaria parasites .…”