Abstract
While conducting pilot studies into the usefulness of fusion to TELSAM polymers as a potential protein crystallization strategy, we observed novel properties in crystals of two TELSAM-target protein fusions, as follows. (i) A TELSAM-target protein fusion can crystallize more rapidly and with greater propensity than the same target protein alone. (ii) TELSAM-target protein fusions can be crystallized at low protein concentrations. This unprecedented observation suggests a route to crystallize proteins that can only be produced in microgram amounts. (iii) The TELSAM polymers themselves need not directly contact one another in the crystal lattice in order to form well-diffracting crystals. This novel observation is important because it suggests that TELSAM may be able to crystallize target proteins too large to allow direct inter-polymer contacts. (iv) Flexible TELSAM-target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. (v) TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts. (vi). Fusion to TELSAM polymers can stabilize weak inter-target protein crystal contacts. We report features of these TELSAM-target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and determine best practices for its use.
Highlights
Atomic-resolution protein structures are essential for structure–function studies, structure-based drug design and biomedical protein engineering
The von Willebrand domain (vWa) domain was chosen because it has been successfully crystallized previously, has a known structure and is only moderately soluble. These properties make the vWa domain an excellent representative target protein to evaluate potential crystallization chaperones. As this was a pilot study, we initially evaluated TEL SAM domain (TELSAM) with target proteins that we reasonably expected would crystallize
54 59 evaluate whether fusion to TELSAM could enhance the properties of vWa domain crystallization relative to the vWa alone
Summary
Atomic-resolution protein structures are essential for structure–function studies, structure-based drug design and biomedical protein engineering. X-ray crystallography remains an important technique to determine atomic-level protein structure, especially of proteins too small for single-particle cryo-electron microscopy. X-ray crystallography provides high-resolution protein structures that can be docked into lower resolution cryo-electron maps. Protein crystals are needed for micro-electron diffraction [1] and time-resolved diffraction using X-ray free-electron lasers [2]. Current protein crystallization methods are successful for only about 10% of all known proteins [3] and constitute a lengthy, laborious and expensive process [4]. Lack of high-resolution structures hampers the structure–function studies of many proteins. There is a critical need for new protein crystallization methods that require less labour, time and resources and that can induce the crystallization of a wider range of proteins. Our ultimate goal is to develop a protein crystallization chaperone that consistently meets the following requirements: (i) is easy to express in Escherichia coli and purify with sufficient yield to screen crystallization conditions, even when fused to target proteins; (ii) enables fused targets to be sufficiently soluble for crystallization screens; (iii) crystallizes in no more than 30 days; (iv) forms crystals that diffract to better than 2 Å with an estimated mosaicity less than 2°; and (v) results in target proteins being well resolved in the crystallographic lattice following molecular replacement or direct phasing
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