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Natalia Oganesyan

Publications and source records attributed to Natalia Oganesyan.

8 recordsLinked to original sources
PubMed2006

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization.

Overexpressed recombinant proteins in bacteria often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or of chaperones. This can be accomplished by growing the bacterial cells in the presence of high salt, sorbitol, and betaine as well as exposing the cells to a heat shock step. Our results suggest that by growing the cells under varied conditions one may be able to express targets as soluble proteins (from previously insoluble targets) and to improve the chances of their crystallization.

Crystallization↗
PubMed2005

Crystal structure of a nicotinate phosphoribosyltransferase from Thermoplasma acidophilum.

We have determined the crystal structure of nicotinate phosphoribosyltransferase from Themoplasma acidophilum (TaNAPRTase). The TaNAPRTase has three domains, an N-terminal domain, a central functional domain, and a unique C-terminal domain. The crystal structure revealed that the functional domain has a type II phosphoribosyltransferase fold that may be a common architecture for both nicotinic acid and quinolinic acid (QA) phosphoribosyltransferases (PRTase) despite low sequence similarity between them. Unlike QAPRTase, TaNAPRTase has a unique extra C-terminal domain containing a zinc knuckle-like motif containing 4 cysteines. The TaNAPRTase forms a trimer of dimers in the crystal. The active site pocket is formed at dimer interfaces. The complex structures with phosphoribosylpyrophosphate (PRPP) and nicotinate mononucleotide (NAMN) showed, surprisingly, that functional residues lining on the active site of TaNAPRTase are quite different from those of QAPRTase, although their substrates are quite similar to each other. The phosphate moiety of PRPP and NAMN is anchored to the phosphate-binding loops formed by backbone amides, as found in many alpha/beta barrel enzymes. The pyrophosphate moiety of PRPP is located at the entrance of the active site pocket, whereas the nicotinate moiety of NAMN is located deep inside. Interestingly, the nicotinate moiety of NAMN is intercalated between highly conserved aromatic residues Tyr(21) and Phe(138). Careful structural analyses combined with other NAPRTase sequence subfamilies reveal that TaNAPRTase represents a unique sequence subfamily of NAPRTase. The structures of TaNAPRTase also provide valuable insight for other sequence subfamilies such as pre-B cell colony-enhancing factor, known to have nicotinamide phosphoribosyltransferase activity.

Amino Acid Sequence↗
PubMed2005

Structural genomics of minimal organisms and protein fold space.

The initial aim of the Berkeley Structural Genomics Center is to obtain a near-complete structural complement of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter fewer than 700 genes. To achieve this goal, the current protein targets have been selected starting with those predicted to be most tractable and likely to yield new structural and functional information. During the past 3 years, the semi-automated structural genomics pipeline has been set up from cloning, expression, purification, and ultimately to structural determination. The results from the pipeline substantially increased the coverage of the protein fold space of M. pneumoniae and M. genitalium. Furthermore, about 1/2 of the structures of 'unique' protein sequences revealed new and novel folds, and over 2/3 of the structures of previously annotated 'hypothetical proteins' inferred their molecular functions.

Bacterial Proteins↗
PubMed2005

On-column protein refolding for crystallization.

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an 'artificial chaperone-assisted refolding' method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.

Chromatography, Affinity↗
PubMed2005

Crystal structure of the "PhoU-like" phosphate uptake regulator from Aquifex aeolicus.

The phoU gene of Aquifex aeolicus encodes a protein called PHOU_AQUAE with sequence similarity to the PhoU protein of Escherichia coli. Despite the fact that there is a large number of family members (more than 300) attributed to almost all known bacteria and despite PHOU_AQUAE's association with the regulation of genes for phosphate metabolism, the nature of its regulatory function is not well understood. Nearly one-half of these PhoU-like proteins, including both PHOU_AQUAE and the one from E. coli, form a subfamily with an apparent dimer structure of two PhoU domains on the basis of their amino acid sequence. The crystal structure of PHOU_AQUAE (a 221-amino-acid protein) reveals two similar coiled-coil PhoU domains, each forming a three-helix bundle. The structures of PHOU_AQUAE proteins from both a soluble fraction and refolded inclusion bodies (at resolutions of 2.8 and 3.2A, respectively) showed no significant differences. The folds of the PhoU domain and Bag domains (for a class of cofactors of the eukaryotic chaperone Hsp70 family) are similar. Accordingly, we propose that gene regulation by PhoU may occur by association of PHOU_AQUAE with the ATPase domain of the histidine kinase PhoR, promoting release of its substrate PhoB. Other proteins that share the PhoU domain fold include the coiled-coil domains of the STAT protein, the ribosome-recycling factor, and structural proteins like spectrin.

Amino Acid Sequence↗
PubMed2004

An automated small-scale protein expression and purification screening provides beneficial information for protein production.

One of the first key steps in structural genomics is high-throughput expression and rapid screening to select highly soluble proteins, the preferred candidates for crystal production. Here we describe the methodology used at the Berkeley Structural Genomics Center (BSGC) for automated parallel expression and small-scale purification of fusion proteins using a 96-well format. Our robotic method includes cell lysis, soluble fraction separation and purification with affinity resins. For detection of His-tagged proteins in the soluble fractions and after affinity resin elution, a dot-blot procedure with an anti-His-antibody is used. The expression level and molecular mass of recombinant proteins are checked by SDS-PAGE. With this approach, we are able to obtain beneficial information to be used for large-scale protein expression and purification.

Automation↗
PubMed2002

The crystal structure of the endothelial protein C receptor and a bound phospholipid.

The endothelial cell protein C receptor (EPCR) shares approximately 20% sequence identity with the major histocompatibility complex class 1/CD1 family of molecules, accelerates the thrombin-thrombomodulin-dependent generation of activated protein C, a natural anticoagulant, binds to activated neutrophils, and can undergo translocation from the plasma membrane to the nucleus. Blocking protein C/activated protein C binding to the receptor inhibits not only protein C activation but the ability of the host to respond appropriately to bacterial challenge, exacerbating both the coagulant and inflammatory responses. To understand how EPCR accomplishes these multiple tasks, we solved the crystal structure of EPCR alone and in complex with the phospholipid binding domain of protein C. The structures were strikingly similar to CD1d. A tightly bound phospholipid resides in the groove typically involved in antigen presentation. The protein C binding site is outside this conserved groove and is distal from the membrane-spanning domain. Extraction of the lipid resulted in loss of protein C binding, which could be restored by lipid reconstitution. CD1d augments the immune response by presenting glycolipid antigens. The EPCR structure is a model for how CD1d binds lipids and further suggests additional potential functions for EPCR in immune regulation, possibly including the anti-phospholipid syndrome.

Animals↗