Solution structure of human sorting nexin 22.

The sorting nexins (SNXs) constitute a large group of PX domain-containing proteins that play critical roles in protein trafficking. We report here the solution structure of human sorting nexin 22 (SNX22). Although SNX22 has <30% sequence identity with any PX domain protein of known structure, it was found to contain the alpha/beta fold and compact structural core characteristic of PX domains. Analysis of the backbone dynamics of SNX22 by NMR relaxation measurements revealed that the two walls of the ligand binding cleft undergo internal motions: on the picosecond timescale for the beta1/beta2 loop and on the micro- to millisecond timescale for the loop between the polyproline motif and helix alpha2. Regions of the SNX22 structure that differ from those of other PX domains include the loop connecting strands beta1 and beta2 and the loop connecting helices alpha1 and alpha2, which appear to be more mobile than corresponding loops in other known structures. The interaction of dibutanoyl-phosphatidylinositol-3-phosphate (dibutanoyl-PtdIns(3)P) with SNX22 was investigated by an NMR titration experiment, which identified the binding site in a basic cleft and indicated that ligand binding leads only to a local structural rearrangement as has been found with other PX domains. Because motions in the loops are damped out when dibutanoyl-PtdIns(3)P binds, entropic effects could contribute to the lower affinity of SNX22 for this ligand compared to other PX domains.

Wheat germ cell-free platform for eukaryotic protein production.

We describe a platform that utilizes wheat germ cell-free technology to produce protein samples for NMR structure determinations. In the first stage, cloned DNA molecules coding for proteins of interest are transcribed and translated on a small scale (25 microL) to determine levels of protein expression and solubility. The amount of protein produced (typically 2-10 microg) is sufficient to be visualized by polyacrylamide gel electrophoresis. The fraction of soluble protein is estimated by comparing gel scans of total protein and soluble protein. Targets that pass this first screen by exhibiting high protein production and solubility move to the second stage. In the second stage, the DNA is transcribed on a larger scale, and labeled proteins are produced by incorporation of [(15)N]-labeled amino acids in a 4 mL translation reaction that typically produces 1-3 mg of protein. The [(15)N]-labeled proteins are screened by (1)H-(15)N correlated NMR spectroscopy to determine whether the protein is a good candidate for solution structure determination. Targets that pass this second screen are then translated in a medium containing amino acids doubly labeled with (15)N and (13)C. We describe the automation of these steps and their application to targets chosen from a variety of eukaryotic genomes: Arabidopsis thaliana, human, mouse, rat, and zebrafish. We present protein yields and costs and compare the wheat germ cell-free approach with alternative methods. Finally, we discuss remaining bottlenecks and approaches to their solution.

Solution structure of Arabidopsis thaliana protein At5g39720.1, a member of the AIG2-like protein family.

The three-dimensional structure of Arabidopsis thaliana protein At5g39720.1 was determined by NMR spectroscopy. It is the first representative structure of Pfam family PF06094, which contains protein sequences similar to that of AIG2, an A. thaliana protein of unknown function induced upon infection by the bacterial pathogen Pseudomonas syringae. The At5g39720.1 structure consists of a five-stranded beta-barrel surrounded by two alpha-helices and a small beta-sheet. A long flexible alpha-helix protrudes from the structure at the C-terminal end. A structural homology search revealed similarity to three members of Pfam family UPF0131. Conservation of residues in a hydrophilic cavity able to bind small ligands in UPF0131 proteins suggests that this may also serve as an active site in AIG2-like proteins.

Wheat germ cell-free expression system for protein production.

The Center for Eukaryotic Structural Genomics, in cooperation with Ehime University and CellFree Sciences, has developed a novel wheat germ cell-free technology for the production of eukaryotic proteins. Protein production and purification are robust and scalable for high-throughput applications. The protocols have been used to express and purify proteins from Arabidopsis thaliana, human, mouse, rat and zebra fish. This unit describes expression and purification protocols for both small-scale testing (microgram) and large-scale production (milligram) of N-His6- and N-GST-tagged proteins. The methods described in this unit can be used to produce both unlabeled and labeled proteins required for structure-based determinations by NMR spectroscopy or X-ray crystallography.