Description

The goal of this work is to obtain high resolution protein X-ray crystal structures of the full-length Taspase1 protein that will facilitate chemical optimization of covalent and non-covalent inhibitors. The structure of the full-length form of Taspase1 and of a complex of the protein with substrate peptide bound will reveal new ligand binding interactions in the catalytic site, which can be exploited for inhibitor design. Subsequently, solving structures of complexes with inhibitors will guide further improvements in inhibitor potency.
The scope of this work initially encompasses collaboration with Beryllium to obtain purified, full-length circularly permuted protein, or potentially with Liang Tongs lab in which work on the 2-chain truncated form of the protein is underway. Crystals containing peptide substrate bound to Taspase1 will most likely be obtained by co-crystallization of peptide substrate with an inactive T234A mutant of Taspase1 in which the threonine catalytic nucleophile has been replaced. But, various other alternative approaches may be tried. For example, a substrate peptide with a cysteine incorporated into the P3 position is efficiently tethered to Cys293 (studies at UCSF) suggesting that the structure of a covalent enzyme-peptide substrate complex could be obtained. Or, purification of enzyme locally at ASU may be required.

A critical bottleneck for protein nanocrystallography is the identification of quality samples for diffraction experiments. While the presence of nanocrystals can be verified by second order nonlinear optics methods like SONICC, these nanocrystals are too small to be directly visualized by optical microscopy and, their small size also prevents high-throughput screening of crystal quality by preliminary X-ray diffraction experiments at conventional home X-ray sources and Synchrotrons. The research team has already identified several potential lead conditions that have produced al promising Taspase1 nanocrystal samples. To greatly accelerate the timeline for the Taspase1 structure determination, we propose to use a high-throughput transmission electron microscopy (TEM) screening strategy (Fig. 1) to rapidly identify which of these initial conditions are able to produce well-diffracting nanocrystals that are suitable for the high-resolution structure determination of Taspase-1. Because this TEM workflow requires very small amounts of sample (approximately 0.1 to 2 L), material can be taken directly from the crystallization plates, which saves valuable time and resources as every potential nanocrystal hit does not need to be scaled up for analysis. This unique strategy combines established negative stain EM procedures (Stevenson, Makhov et al. 2014) to rapidly narrow down the pool of crystallization conditions along with the recently developed cryo-electron diffraction technique, MicroED (Nannenga and Gonen 2016), to identify only those conditions which form quality nanocrystals capable of diffracting to high resolution.
StatusActive
Effective start/end date4/1/1612/31/19

Funding

  • HHS: National Institutes of Health (NIH): $27,138,473.00

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Nanocrystals
Membrane Proteins
Peptides
Crystallization
Substrates
Proteins
Screening
Throughput
Transmission electron microscopy
Beryllium
X rays
Nonlinear optics
Crystals
Nucleophiles
Enzymes
Threonine
Synchrotrons
Electron diffraction
Optical microscopy
Purification