Protein crystallization in vivo
Introduction
The crystallization of proteins in vitro is a subject of immense practical importance, partly because of the vital role played by protein crystallography in modern structural molecular biology. Thus, considerable effort has been devoted to understanding how to crystallize proteins in the laboratory [1•]. In the last decade, colloid scientists have contributed significantly to this enterprise [2•]. By treating globular proteins in a coarse-grained manner, it turns out that certain regularities, such as the existence of an optimal ‘crystallization window', can be rationalized [3]. Initially, the simplest possible model was used—complex protein molecules were reduced to perfect hard spheres with isotropic, short-range inter-particle attraction. Subsequently, more realistic models begin to appear. For example, the ‘stickiness’ can occur in patches (either regular [4] or random [5]). On the other hand, the study of proteins can give new insights into problems in colloid science, such as the existence of finite, equilibrium clusters in charged, attractive particle systems [6]. Thus, the intellectual traffic between the study of protein solutions and colloidal suspensions has proved fruitful for both sides.
Protein crystals also occur in vivo. This phenomenon is, of course, one example of biological self-assembly. Some other examples include the formation of viral capsids [7] and multi-protein complexes such as the ribosome [8]. Compared to these more celebrated examples, in vivo protein crystallization has been relatively neglected: we know of only one attempt to provide a general survey (see the last chapter in Ref. [1•]). One of the reasons for this relative neglect is that in vivo protein crystallization is perceived to be an atypical behaviour, and for good reason: protein aggregation and crystallization would normally be expected to be deleterious to the cell. Indeed, the difficulty of crystallizing proteins in vitro may reflect precisely the ‘negative selection’ that has occurred in vivo over evolutionary time scales against easily crystallizable variants [9•].
The purpose of this article is to provide a review of in vivo protein crystallization from the perspective of colloid science. Unlike other articles in this journal, many of the references will necessarily not be ‘current’ in the chronological sense—some of the first examples of in vivo protein crystals go back nearly a century or more. However, we hope that the topic is of significant current interest. Understanding in vivo protein crystallization, and perhaps how cells have evolved to avoid it in the main, may be an area that is ripe for contributions from colloid scientists. We organize the examples reviewed according to the putative biological functions of the protein crystals in vivo. In the concluding discussion we seek to draw together some elements of commonality between different kinds of in vivo protein crystals, between in vivo and in vitro protein crystallization, as well as point to some of the interesting differences.
Section snippets
Protein storage
Perhaps the most ‘obvious’ use for protein crystals in vivo is for storage, either temporarily, with a view to future utilization or excretion, or as a means of permanent sequestration. This section gathers examples of this kind.
Encapsulation
Some particularly interesting examples of protein crystallization occur in certain genera of insect viruses, namely cytoplasmic and nuclear polyhedrosis viruses, granulosis viruses and entomopoxviruses [35••]. All these viruses coopt the infected cells to express large quantities of proteins (polyhedrin, granulin and spheroidin, respectively, in the above classes) in the late stages of infection. (Indeed, the baculovirus protein expression systems, one of the most popular eukaryotic
Solid-state catalysts: peroxisome enzymes
Peroxisomes are membrane-bound organelles found in eukaryotic cells that are responsible for a variety of chemical processes, such as the breakdown of lipids and alcohol, that are catalysed by enzymes, such as catalase, urate oxidase and alcohol oxidase, that are typically assembled into regular crystals. These reactions are usually oxidative in nature and often involve unpleasant chemical species such as H2O2, hence the need for confinement in a specific organelle. The crystals can be both
Woronin bodies
In filamentous fungi, cellular compartments are connected by septal pores that allow trafficking between cells. When a cell is damaged, the septal pore is sealed by Woronin bodies, preventing cytoplasmic bleeding. Each Woronin body is a hexagonal platelet ≈5 μm across, and consists of a proteinaceous core that is primarily crystalline HEX-1 surrounded by a membrane [43]. The HEX-1 protein contains a peroxisomal targeting sequence that causes it to be trafficked to the organelles, and thus the
Disease induced crystallization
For most proteins, crystallization within the cellular environment is to be avoided, and crystallization is a sign of dysfunction. For example, we know of two diseases that are directly caused by protein crystallization. Firstly, hemoglobin C disease is associated with a mutant form of hemoglobin, in which a glutamate is replaced by lysine. Crystallization of this hemoglobin C can occur in the red blood cells of people who are homozygous for the gene encoding this mutation (CC) or who have a
Crystallization of larger proteinaceous structures
There are also many examples of larger proteinaceous bodies forming (para-)crystalline structures in vivo. For example, in cells containing large numbers of icosahedral viral particles, crystals are frequently seen to occur. The resulting iridescence due to Bragg scattering of visible light lies behind the naming of the iridovirus family [35••]. It is not clear whether this behaviour has a functional purpose—perhaps the formation of a crystal maximizes the room for more viruses to be produced,
Conclusion
From even such a short survey—we could have provided many more examples—it is clear that protein crystallization in vivo, while not necessarily ubiquitous, is not nearly as esoteric a phenomenon in biology as one might have thought. In our selection, we have dwelt on those cases that are the most well-characterized, and the function the most clear. However, in a number of instances, particularly those associated with storage, although it is clear why a dense state is favoured, it is less clear
Acknowledgements
We thank Rod Casey and Clare Mills for introducing us to seed storage proteins, Nick Read for educating us about Woronin bodies, Ard Louis, Robert Possee and Michele Vendruscolo for stimulating discussions, and Linda Sperling for provision of original micrographs and for helpful comments on the manuscript.
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