Structural Genomics of Pathogenic Protozoa (SGPP)

(Peter Myler)

Leishmania offer a unique opportunity to clone and express protein variants, as there are 10 species of these parasites with from 60-90% identity to L. major.  We amplified 141 total targets from at least one other Leishmania species using primers designed from L. major genome.  For at least 8 of these targets, a clone obtained from a non-L. major genome was able to progress further down the pipeline than the L. major cognate, including one case where the structure was solved.  The use of multiple species is a powerful means to increase the chances of getting a structure from a high-value target.

 

(David Kim, Dylan Chivian, Lars Marlstrom, David Baker)

We have utilized an iterative domain-identification procedure called "Ginzu" to predict protein domains in nearly all of the SGPP targets.  9,055 targets were parsed into 14,515 putative domains with an average length of 259 residues.  This accounts for 23,054 "chunks," defined as any combination of single or consecutive domains.  Many chunks have now entered the SGPP pipeline.  Current applications include using Ginzu to identify domains from yeast two-hybrid P. falciparum pairs.  We anticipate that a significant number of full-length proteins that are insoluble will prove to be soluble when they are expressed as chunks.

 

(Doug LaCount, Marissa Vignali, Lori Schoenfeld, Stan Fields and collaborators)

A large-scale yeast two-hybrid assay was conducted to identify interacting proteins from the intraerythrocytic phase of the P. falciparum life cycle.  In conjunction with collaborators at Prolexys Pharmaceuticals, we obtained 4,878 different putative interactions involving 1,513 proteins.  We are currently assessing our data using two-hybrid criteria to weed out false positives, and searching amongst the strong interactions for biological relevance.  We have supplied the co-expression group with a list of some of our strongest interacting proteins as candidates for their project (see below).

 

(Eric Phizicky, Beth Grayhack, Andrei Alexandrov)

A successful and highly reproducible method for cloning protein pairs on stable tandem plasmids and expressing them as soluble complexes was developed (see Alexandrov A. et al., Mol Cell Proteomics, 2004 Jul 7 [Epub ahead of print]).  We expect to increase the chances of a single insoluble protein to be solubly expressed in a complex, and ultimately to contribute to the increase in number of protein complexes in the Protein Data Bank.  We have initiated the cloning of some of the strongest interacting protein pairs identified by the P. falciparum yeast two-hybrid assay above, as well as work on a list of "interactomes"—genes in L. major with homology to yeast genes whose protein products are known to interact.

 

(Mark Dumont)

L. major targets with two or more predicted transmembrane regions were selected.  A set of four vectors was developed for the expression, secretion and purification of these targets in either E. coli or the methylotropic yeast Pichia pastoris.  In E. coli, 23% of the targets cloned into a vector containing a PelB secretion signal showed detectable expression by Coomassie staining as compared to only 7% cloned into a vector with no signal sequence. In total, six predicted transmembrane ORFs from L. major have now been purified and sent for crystallization trials.  Expression and purity has proved to be more difficult in Pichia, leading us to primarily focus on pushing targets through E. coli expression at this time.

 

(Mark Dumont and collaborators)

P. falciparum coding sequence has an A + T content of approximately 75%, which has been problematic for expression in most of the commonly used expression systems.  T. thermophila has a similar A + T content and may have additional advantages in expressing proteins from a fellow protozoan.  We have constructed a series of vectors compatible with T. thermophila that utilize high-throughput, ligation-independent cloning to insert P. falciparum genes.  We will use three different vector constructs: one for soluble proteins, one for membrane proteins using secretion signals from the ORF, and another for membrane proteins that attaches a heterologous secretion signal.  Cloning of test ORFs into these vectors is underway.

 

(Mark Sullivan)

Creating antibody-protein complexes can be an effective way to crystallize a recalcitrant target.  We have utilized phage display of antibody libraries to enrich on 16 target proteins and have succeeded with 12 of these. Thus far, we have been able to isolate and send one of these complexes for crystallization.  The remaining 11 have been expressed and are undergoing scale-up and evaluation.

 

(Erkang Fan, Christophe Verlinde, Joe Luft, Stacey Gulde, George De Titta)

We have developed the capability of synthesizing 44 synthetic compounds at the rate of 8 per month.  These compounds will be screened with protein targets in a 1536-well format using the standard crystallization buffers designed by HWI.  50 target proteins that were selected for their inability to crystallize and/or diffract were recently sent to HWI for screening with these 44 compounds.

 

(Erkang Fan, Yuko Ogata and co-workers)

Using an affinity assay to screen for high affinity compounds will allow us to search through a large chemical space for potential co-crystallants.  We have validated Lab-on-Valve (LOV) as an automatable setup for running FAC screening (Ogata et al, Anal Biochem, 2004, in press).  Our current assay includes ~40 compounds including common co-factors, hydrophobic dipeptides and some synthetic co-crystallants.  Through manual screening, we have already observed one L. major protein with a high affinity to 5'-TMP.  Because this target has no annotated function, validation of its specific binding to this cofactor may elucidate its function.

 

(Deirdre Meldrum, Shawn McGuire, Mark Holl, Larry De Soto, Wim Hol)

 A robotics system for protein crystal growth in plastic capillaries has three primary advantages:  optimization of crystal growth by free-interface diffusion through the capillaries, use of low protein and precipitant volumes, and the full automation of the entire process from protein solution to mounted crystals, imaging, crystal centering and X-ray data collection.  We are awaiting the delivery of several new developments including the modification of the ACAPELLA loading hopper to automate the handling of plastic capillaries.  Currently in development is the upgrade of the piezoelectric reagent dispensers for the optimal dispensing of very small volumes of proteins and reagents.

 

(Ethan Merritt, Martin Berg and co-workers)

Protein crystals are cooled to liquid nitrogen temperatures to protect them from ionizing damage during X-ray diffraction.  Cooling often produces lattice disorder, which later can be reduced by subsequent "annealing," i.e., raising and lowering the temperature of the crystal as well as varying the solvent content of the crystal.  Our automated system will include an X-ray diffractometer with a motorized goniometer, a liquid nitrogen delivery system, a dewar with an automated system for controlling its liquid nitrogen level, a tabletop robot, an annealing station, and a humidity-controlled "healing" chamber.  All will operate under the commands of a central controller that responds to commands issued by a human operator via a graphical user interface (GUI).