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1.  DisMeta – a Meta Server for Construct Design and Optimization 
Summary
Intrinsically disordered or unstructured regions in proteins are both common and biologically important, particularly in regulation, signaling and modulating intermolecular recognition processes. From a practical point of view, however, such disordered regions often can pose significant challenges for crystallization. Disordered regions are also detrimental to NMR spectral quality, complicating the analysis of resonance assignments and three-dimensional protein structures by NMR methods. The DisMeta Server has been used by Northeastern Structural Genomics Consortium (NESG) as a primary tool for construct design and optimization in preparing samples for both NMR and crystallization studies. It is a meta-server that generates a consensus analysis of eight different sequence-based disorder predictors to identify regions that are likely to be disordered. DisMeta also identifies predicted secretion signal peptides, trans-membrane segments, and low complexity regions. Identification of disordered regions, by either experimental or computational methods, is an important step in the NESG structure production pipeline, allowing the rational design of protein constructs that have improved expression and solubility, improved crystallization, and better quality NMR spectra.
doi:10.1007/978-1-62703-691-7_1
PMCID: PMC4115584  PMID: 24203321
intrinsically disorder protein prediction; construct design; construct optimization; hydrogen-deuterium exchange with mass spectrometry (HDX-MS)
2.  The High-Throughput Protein Sample Production Platform of the Northeast Structural Genomics Consortium 
Journal of structural biology  2010;172(1):21-33.
We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (> 97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as > 26,000 constructs. Over the past nine years, more than 16,000 of these expressed protein, and more than 4,400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last five years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.
doi:10.1016/j.jsb.2010.07.011
PMCID: PMC4110633  PMID: 20688167
Structural Genomics; High throughput protein production; Construct optimization; Disorder prediction; Ligation independent cloning; Multiple Displacement Amplification; Laboratory Information Management System; Protein Structure Initiative; NMR; X-ray crystallography; T7 Escherichia Coli expression system; Wheat Germ Cell Free; NMR microprobe screening; Parallel protein purification; 6X-His tag; HDX-MS
3.  Preparation of Protein Samples for NMR Structure, Function, and Small Molecule Screening Studies 
Methods in enzymology  2011;493:21-60.
In this chapter, we concentrate on the production of high quality protein samples for NMR studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium, and outline our high-throughput strategies for producing high quality protein samples for nuclear magnetic resonance (NMR) studies. Our strategy is based on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6X-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (> 97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5,000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this paper describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening and structural genomics research.
doi:10.1016/B978-0-12-381274-2.00002-9
PMCID: PMC4110644  PMID: 21371586
Structural Genomics; High throughput protein production; Construct optimization; Disorder prediction; Ligation independent cloning; Multiple Displacement Amplification; Laboratory Information Management System; Protein Structure Initiative; NMR; T7 Escherichia coli expression system; Wheat Germ Cell Free; NMR microprobe screening; Parallel protein purification; 6X-His tag; HDX-MS; Total gene synthesis; condensed single protein production
4.  Human retinoblastoma binding protein 9, a serine hydrolase implicated in pancreatic cancers 
Protein and peptide letters  2012;19(2):194-197.
Human retinoblastoma binding protein 9 (RBBP9) is an interacting partner of the retinoblastoma susceptibility protein (Rb). RBBP9 is a tumor-associated protein required for pancreatic neoplasia, affects cell cycle control, and is involved in the TGF-β signalling pathway. Sequence analysis suggests that RBBP9 belongs to the α/β hydrolase superfamily of enzymes. The serine hydrolase activity of RBBP9 is required for development of pancreatic carcinomas in part by inhibiting TGF-β antiproliferative signaling through suppressing Smad2/3 phosphorylation. The crystal structure of human RBBP9 confirms the α/β hydrolase fold, with a six-stranded parallel β-sheet flanked by α helixes. The structure of RBBP9 resembles that of the YdeN protein from Bacillus subtilis, which is suggested to have carboxylesterase activity. RBBP9 contains a Ser75-His165-Asp138 catalytic triad, situated in a prominent pocket on the surface of the protein. The side chains of the LxCxE sequence motif that is important for interaction with Rb is mostly buried in the structure. Structure-function studies of RBBP9 suggest possible routes for novel cancer drug discovery programs.
PMCID: PMC3677193  PMID: 21933118
α/β hydrolase; pancreatic cancer; protein structure; structural genomics; RBBP9
5.  UNC119 is required for G protein trafficking in sensory neurons 
Nature neuroscience  2011;14(7):874-880.
SUMMARY
UNC119 is widely expressed among vertebrates and invertebrates. Here we report that UNC119 recognized the acylated N-terminus of the rod photoreceptor transducin α-subunit (Tα) as well as C. elegans G proteins Odr-3 and Gpa-13. The crystal structure of human UNC119 at 1.95 Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. Co-crystallization of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound TαGTP inhibiting its GTPase activity, thereby providing a stable UNC119-TαGTP complex that is capable of diffusing from the inner segment back to the outer segment following light-induced translocation. UNC119 deletion in both mouse and C. elegans lead to G protein mislocalization. These results establish UNC119 as a novel Gα-subunit cofactor that is essential for G-protein trafficking in sensory cilia.
doi:10.1038/nn.2835
PMCID: PMC3178889  PMID: 21642972
6.  RPF: a quality assessment tool for protein NMR structures 
Nucleic Acids Research  2012;40(Web Server issue):W542-W546.
We describe the RPF web server, a quality assessment tool for protein NMR structures. The RPF server measures the ‘goodness-of-fit’ of the 3D structure with NMR chemical shift and unassigned NOESY data, and calculates a discrimination power (DP) score, which estimates the differences between the fits of the query structures and random coil structures to these experimental data. The DP-score is an accuracy predictor of the query structure. The RPF server also maps local structure quality measures onto the 3D structure using an online molecular viewer, and onto the NMR spectra, allowing refinement of the structure and/or NOESY peak list data. The RPF server is available at: http://nmr.cabm.rutgers.edu/rpf.
doi:10.1093/nar/gks373
PMCID: PMC3394279  PMID: 22570414
8.  Outcome of a Workshop on Applications of Protein Models in Biomedical Research 
Summary
We describe the proceedings and conclusions from a “Workshop on Applications of Protein Models in Biomedical Research” that was held at University of California at San Francisco on 11 and 12 July, 2008. At the workshop, international scientists involved with structure modeling explored (i) how models are currently used in biomedical research, (ii) what the requirements and challenges for different applications are, and (iii) how the interaction between the computational and experimental research communities could be strengthened to advance the field.
doi:10.1016/j.str.2008.12.014
PMCID: PMC2739730  PMID: 19217386

Results 1-8 (8)