Structure of thymidylate kinase from Ehrlichia chaffeensis

A 2.15 Å resolution apo structure of thymidylate kinase from E. chaffeensis is reported

High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

An overview of the standard SSGCID protein-purification protocol is given and success rates and cleavage alternatives are discussed

Structure of the cystathionine γ-synthase MetB from Mycobacterium ulcerans

Cystathionine γ-synthase (CGS) is a transferase that catalyzes the reaction between O 4-succinyl-l-homoserine and l-cysteine to produce l-­cystathionine and succinate. The crystal structure of CGS from M. ulcerans is presented covalently linked to the cofactor pyridoxal phosphate (PLP). A second structure contains PLP as well as a highly ordered HEPES molecule in the active site acting as a pseudo-ligand. This is the first structure ever reported from the pathogen M. ulcerans

Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region.Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 A resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs.The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics

Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins.

Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli) appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2), proline, xylitol, NDSB 201, CTAB and K(2)PO(4)) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher

A Suite of Engineered GFP Molecules for Oligomeric Scaffolding

Applications ranging from synthetic biology to protein crystallization could be advanced by facile systems for connecting multiple proteins together in predefined spatial relationships. One approach to this goal is to engineer many distinct assembly forms of a single carrier protein or scaffold, to which other proteins of interest can then be readily attached. In this work we chose GFP as a scaffold and engineered many alternative oligomeric forms, driven by either specific disulfide bond formation or metal ion addition. We generated a wide range of spatial arrangements of GFP subunits from 11 different oligomeric variants, and determined their X-ray structures in a total of 33 distinct crystal forms. Some of the oligomeric GFP variants show geometric polymorphism depending on conditions, while others show considerable geometric rigidity. Potential future applications of this system are discussed

SDS-PAGE gels from the screen of <i>T. gondii</i> Hsp20.

<p>(A) Presented is one of the six SDS-PAGE gels from the primary screening experiments of Hsp20 from <i>Toxoplasma gondii.</i> Six screening gels are performed per protein to screen all 144 unique cell lysis conditions, 24 conditions per gel. Each gel is run with the protein-lysed apo (without additives) on the far left side of the gel. “Std. Total” is total cell lysate, lysed without additives. “Std. Soluble” is the soluble fraction of the non-additive lysed cells. The lanes between ‘A1’ and ‘B12’ are the 24 conditions screened on this gel. The expected molecular weight of the recombinant protein, <i>Toxoplasma gondii</i> Hsp20, is indicated with the arrow. The conditions that appeared to increase solubility and were subsequently re-screened are LDAO (A2), L-Arginine (B2), L-Proline (B3), Glycine Betaine (B4), Mannitol (B7), Trehalose (B8), NDSB 195 (B11), and Trimethylamine N-Oxide (B12). (B) SDS-PAGE gel image shows the individual secondary screen for the <i>Toxoplasma gondii</i> Hsp20 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052482#pone-0052482-g001" target="_blank">figure 1A</a>. The <i>Toxoplasma</i> Hsp20 protein has an ‘Apo’ control that was lysed without any additives present. Additive conditions are in the lane to the right of each Apo. Lanes marked ‘T’ are the total cell lysate, lanes marked ‘S’ are the soluble fraction for each condition. The overexpressed protein band at ∼28 kDa is the protein of interest. Conditions B8 (Trehalose) and B11 (NDSB 195) proved to be the best in solubilizing the protein, where close to 100% of the protein was present in the soluble fraction when compared to the total cell lysate, as opposed to conditions B2–B7 where there is a clear distinction between the relative amount of recombinant protein present in the soluble fraction compared to the total fraction.</p