Abstracts D1-D4

Category D.  Protein Structure & Function

 

D1.  A Reclassification of Outer Membrane β-Barrels

Meghan Franklin1, Ryan Feehan2, Joanna Slusky1,2

1Center for Computational Biology, University of Kansas, Lawrence, KS, USA;
2Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA

The outer membrane proteins of Gram-negative bacteria are nearly all β-barrels. They perform a diverse range of functions. They are especially important as antibacterial targets because of their ability to transport metal-containing siderophores, convey antibiotic resistance, and to facilitate biofilm formation. Structurally, these barrels differ from soluble β-barrels and a cohesive functional, structural and evolutionary classification has been difficult due to the lack of structural information. Here we find structural categories for these barrels that correlate with function. We also find evolutionary relationships between the barrels which tell a story of how they evolved.

In this work we used the HMM profile of 99 non-homologous β-barrels with crystal structures to construct a network indicating the relatedness between them. We analyzed structural hallmarks for the 5 classes identified. While most β-barrels exhibit a linear relationship between number of strands per barrel and barrel radius, the class of efflux pumps are disproportionately wide and unusually tilted. The assembly barrels, such as BamA, and the adhesins have a narrow distribution of radius and heights, while the hemolysins are longer than other barrels with the same number of strands. Finally, there is a small group of OMPs with no immediate relationships; as more crystal structures are elucidated, these may develop connections to existing classes or into additional classes.

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D2.  PvdF as Potential Novel Transformylase from Pseudomonas aeruginosa

Nikola Kenjic, Audrey L.Lamb

Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA

Siderophores are bioactive peptides responsible for iron scavenging by bacteria and fungi in iron limiting environments. In the opportunistic pathogen Pseudomonas aeruginosa, pyoverdin is just one of the siderophores made. The enzymatic biosynthesis of these molecules is characterized by “assembly line” production by nonribosomal peptide synthetases (NRPS). One of the amino acids incorporated into pyoverdin is an ornithine derivative that has been modified by the ornithine hydroxylase PvdA, and PvdF. Based on the gene context, it has been proposed that PvdF catalyzes the conversion of hydroxyornithine to formyl - hydroxyornithine using N10-formyltetrahydrofolate (N10-THF) as the formyl donor. Here we demonstrate that a more stable analogue of N10 formyl -THF, fDDf (5,8 dideazafolate ) is turned over enzymatically, as measured by DDF absorbance at 295nm. Full enzyme kinetics for both hydroxyornithine and fDDF, as varied substrates, is presented.   Currently we are in the process of confirming formyl transfer to the product. Improvements in protein purification allowed for easier PvdF crystallization. We have native PvdF crystals that diffract to 2.3Å. Furthermore, we have identified conditions for production of Se-Met variant crystals for multiwavelength anomalous dispersion experiments. These results will be used as the basis for determination of the structure function relationships for this yet uncharacterized class of enzymes.

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D3.  The University of Kansas Protein Structure Laboratory

Nurjahan Mehzabeen and Scott Lovell

Protein Structure Laboratory, Del Shankel Structural Biology Center, University of Kansas, Lawrence, KS, USA

The Center of Biomedical Research Excellence in Protein Structure and Function (COBRE-PSF) at The University of Kansas University supports health-related basic research efforts to obtain structural/functional information for proteins.  One of the three core laboratories at the COBRE-PSF, the Protein Structure Laboratory (PSL), collaborates with investigators from various institutions in an effort to obtain the 3-dimensional structures of proteins using X-ray crystallography.  The capabilities and infrastructure of the PSL are presented here along with examples of collaborative projects that have been completed.  The results from these projects highlight the importance of obtaining structural information to provide mechanistic/functional insight for particular proteins and demonstrate the significance of structural biology to facilitate and support drug discovery efforts.

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D4.  Differential Photosensitivity of IgG1 in Solutions Containing Various Salts

Natalia Subelz1, John Wang2 and Christian Schӧneich1

1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, USA
2Late Stage Pharmaceutical Development Department, Genentech Inc., S. San Francisco, CA, USA

Light sensitivity of pharmaceutical proteins depends on protein nature and additional factors as presence of excipients. Most commons used excipients in protein formulations are salts, which can function as kosmotropes, stabilizing protein structure and promoting salting-out or chaotropes, destabilizing protein structure. Until now, different effect of chaotropes and kosmotropes on protein stability during light irradiation is unknown; specifically we want to focus in the dormation of D-amino acids. For this purpose, we studied the effect of different Hofmeister series ions as excipients on the photostability of a monoclonal antibody (IgG1). Solutions of antibody (5mg/mL) were prepared in argon saturated phosphate buffer (20 mM, pH 5.7) in the presence of 50 mM of selected group of kosmotropes (NH4Cl and Na2CO3) and chaotropes (GndHCl and NaOCl4), compared to a NaCl. Solutions were then irradiated in Rayonet UV irradiator, equipped with four lamps emitting at λmax= 254 nm, during 5 to 30 min. Monomer loss, degraded fractions and aggregates were detected by size exclusion chromatography and SDS-PAGE. Crosslinks and oxidation protein products were identified by HPLC-MS-MS analysis. D-amino acid formation was studied by reverse phase separation in HPLC. Evidence of carbon center radical was performed by covalent deuterium incorporation during IgG1 photoirradiation in D2O. According to our results, we propose a different mechanistic excipient effect on photosensitivity of IgG1. Further studies to identification of other products will be performed.

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