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David A Snyder,
James M Aramini,
Bomina Yu,
Yuanpeng J Huang,
Rong Xiao,
John R Cort, Ritu Shastry,
Li-Chung Ma,
Jinfeng Liu,
Burkhard Rost,
Thomas B Acton,
Michael A Kennedy,
Gaetano T Montelione
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ABSTRACT: The ribosome consists of small and large subunits each composed of dozens of proteins and RNA molecules. However, the functions of many of the individual protomers within the ribosome are still unknown. In this article, we describe the solution NMR structure of the ribosomal protein RP-L35Ae from the archaeon Pyrococcus furiosus. RP-L35Ae is buried within the large subunit of the ribosome and belongs to Pfam protein domain family PF01247, which is highly conserved in eukaryotes, present in a few archaeal genomes, but absent in bacteria. The protein adopts a six-stranded anti-parallel β-barrel analogous to the "tRNA binding motif" fold. The structure of the P. furiosus RP-L35Ae presented in this article constitutes the first structural representative from this protein domain family.
Proteins Structure Function and Bioinformatics 03/2012; 80(7):1901-6. · 3.39 Impact Factor
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Farhad Forouhar,
Nabila Saadat,
Munif Hussain,
Jayaraman Seetharaman,
Insun Lee,
Haleema Janjua,
Rong Xiao, Ritu Shastry,
Thomas B Acton,
Gaetano T Montelione,
Liang Tong
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ABSTRACT: ATP pyrophosphatases (ATP PPases) are widely distributed in archaea and eukaryotes. They share an HUP domain at the N-terminus with a conserved PP-motif that interacts with the phosphates of ATP. The PF0828 protein from Pyrococcus furiosus is a member of the ATP PPase superfamily and it also has a 100-residue C-terminal extension that contains a strictly conserved EGG(E/D)xE(T/S) motif, which has been named the EGT-motif. Here, crystal structures of PF0828 alone and in complex with ATP or AMP are reported. The HUP domain contains a central five-stranded β-sheet that is surrounded by four helices, as in other related structures. The C-terminal extension forms a separate domain, named the EGT domain, which makes tight interactions with the HUP domain, bringing the EGT-motif near to the PP-motif and defining the putative active site of PF0828. Both motifs interact with the phosphate groups of ATP. A loop in the HUP domain undergoes a large conformational change to recognize the adenine base of ATP. In solution and in the crystal PF0828 is a dimer formed by the side-by-side arrangement of the HUP domains of the two monomers. The putative active site is located far from the dimer interface.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2011; 67(Pt 11):1323-7. · 0.51 Impact Factor
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Thomas B Acton,
Rong Xiao,
Stephen Anderson,
James Aramini,
William A Buchwald,
Colleen Ciccosanti,
Ken Conover,
John Everett,
Keith Hamilton,
Yuanpeng Janet Huang, [......],
Paolo Rossi,
Seema Sahdev, Ritu Shastry,
G V T Swapna,
Yeufeng Tang,
Saichiu Tong,
Dongyan Wang,
Huang Wang,
Li Zhao,
Gaetano T Montelione
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ABSTRACT: In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (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 NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-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 6×-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 5000 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 chapter 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.
Methods in enzymology 01/2011; 493:21-60. · 1.90 Impact Factor
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Rong Xiao,
Stephen Anderson,
James Aramini,
Rachel Belote,
William A Buchwald,
Colleen Ciccosanti,
Ken Conover,
John K Everett,
Keith Hamilton,
Yuanpeng Janet Huang, [......],
Seema Sahdev,
Seema Sharma, Ritu Shastry,
G V T Swapna,
Saichu N Tong,
Dongyan Wang,
Huang Wang,
Li Zhao,
Gaetano T Montelione,
Thomas B Acton
[show abstract]
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ABSTRACT: 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 10 years, more than 16,000 of these expressed protein, and more than 4400 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 5 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.
Journal of Structural Biology 10/2010; 172(1):21-33. · 3.41 Impact Factor
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ABSTRACT: Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT((R)) SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354-359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two (15)N-, (13)C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline.
Journal of Structural and Functional Genomics 09/2010; 11(3):201-9.
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Susanne Gräslund,
Pär Nordlund,
Johan Weigelt,
B Martin Hallberg,
James Bray,
Opher Gileadi,
Stefan Knapp,
Udo Oppermann,
Cheryl Arrowsmith,
Raymond Hui, [......],
Mei Jiang,
Gaetano T Montelione,
David I Stuart,
Raymond J Owens,
Susan Daenke,
Anja Schütz,
Udo Heinemann,
Shigeyuki Yokoyama,
Konrad Büssow,
Kristin C Gunsalus
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ABSTRACT: In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
Nature Methods 03/2008; 5(2):135-46. · 19.28 Impact Factor
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Susanne Gr|[auml]|slund,
P|[auml]|r Nordlund,
Johan Weigelt,
James Bray,
Opher Gileadi,
Stefan Knapp,
Udo Oppermann,
Cheryl Arrowsmith,
Raymond Hui,
Jinrong Ming, [......],
Mei Jiang,
Gaetano T Montelione,
David I Stuart,
Raymond J Owens,
Susan Daenke,
Anja Sch|[uuml]|tz,
Udo Heinemann,
Shigeyuki Yokoyama,
Konrad B|[uuml]|ssow,
Kristin C Gunsalus
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ABSTRACT: NOTE: In the version of this Review initially published, an author (B. Martin Hallberg) was left off of the author list. This information has been
added to the HTML and PDF versions of the Review.
Nature Methods 01/2008; 5(2):135-146. · 19.28 Impact Factor
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James M Aramini,
Yuanpeng J Huang,
G V T Swapna,
John R Cort,
P K Rajan,
Rong Xiao, Ritu Shastry,
Thomas B Acton,
Jinfeng Liu,
Burkhard Rost,
Michael A Kennedy,
Gaetano T Montelione
Proteins Structure Function and Bioinformatics 09/2007; 68(3):789-95. · 3.39 Impact Factor
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Journal of Biomolecular NMR 12/2005; 33(3):197. · 3.61 Impact Factor
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Thomas B Acton,
Kristin C Gunsalus,
Rong Xiao,
Li Chung Ma,
James Aramini,
Michael C Baran,
Yi-Wen Chiang,
Teresa Climent,
Bonnie Cooper,
Natalia G Denissova, [......],
Paranji K Rajan, Ritu Shastry,
Liang-Yu Shih,
G V T Swapna,
Michael Wilson,
Margaret Wu,
Mark Gerstein,
Masayori Inouye,
John F Hunt,
Gaetano T Montelione
[show abstract]
[hide abstract]
ABSTRACT: In this chapter 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 using Escherichia coli host vectors. The platform is centered on 6X-His affinity-tagged protein constructs, allowing for a similar purification procedure for most targets, and the implementation of high-throughput parallel methods. In most cases, these affinity-purified proteins are sufficiently homogeneous that a single subsequent gel filtration chromatography step is adequate to produce protein preparations that are greater than 98% pure. Using this platform, over 1000 different proteins have been cloned, expressed, and purified in tens of milligram quantities over the last 36-month period (see Summary Statistics for All Targets, ). Our experience using a hierarchical multiplex expression and purification strategy, also described in this chapter, has allowed us to achieve success in producing not only protein samples but also many three-dimensional structures. As of December 2004, the NESG Consortium has deposited over 145 new protein structures to the Protein Data Bank (PDB); about two-thirds of these protein samples were produced by the NESG Protein Production Facility described here. The methods described here have proven effective in producing quality samples of both eukaryotic and prokaryotic proteins. These improved robotic and?or parallel cloning, expression, protein production, and biophysical screening technologies will be of broad value to the structural biology, functional proteomics, and structural genomics communities.
Methods in Enzymology 02/2005; 394:210-43. · 2.04 Impact Factor
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[show abstract]
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ABSTRACT: The isc and suf operons in Escherichia coli represent alternative genetic systems optimized to mediate the essential metabolic process of iron-sulfur cluster (Fe-S) assembly under basal or oxidative-stress conditions, respectively. Some of the proteins in these two operons share strong sequence homology, e.g. the cysteine desulfurases IscS and SufS, and presumably play the same role in the oxygen-sensitive assembly process. However, other proteins in these operons share no significant homology and occur in a mutually exclusive manner in Fe-S assembly operons in other organisms (e.g. IscU and SufE). These latter proteins presumably play distinct roles adapted to the different assembly mechanisms used by the two systems. IscU has three invariant cysteine residues that function as a template for Fe-S assembly while accepting a sulfur atom from IscS. SufE, in contrast, does not function as an Fe-S assembly template but has been suggested to function as a shuttle protein that uses a persulfide linkage to a single invariant cysteine residue to transfer a sulfur atom from SufS to an alternative Fe-S assembly template. Here, we present and analyze the 2.0A crystal structure of E.coli SufE. The structure shows that the persulfide-forming cysteine occurs at the tip of a loop with elevated B-factors, where its side-chain is buried from solvent exposure in a hydrophobic cavity located beneath a highly conserved surface. Despite the lack of sequence homology, the core of SufE shows strong structural similarity to IscU, and the sulfur-acceptor site in SufE coincides with the location of the cysteine residues mediating Fe-S cluster assembly in IscU. Thus, a conserved core structure is implicated in mediating the interactions of both SufE and IscU with the mutually homologous cysteine desulfurase enzymes present in their respective operons. A similar core structure is observed in a domain found in a variety of Fe-S cluster containing flavoenzymes including xanthine dehydrogenase, where it also mediates interdomain interactions. Therefore, the core fold of SufE/IscU has been adapted to mediate interdomain interactions in diverse redox protein systems in the course of evolution.
Journal of Molecular Biology 12/2004; 344(2):549-65. · 4.00 Impact Factor
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ABSTRACT: IscU is a highly conserved protein that serves as the scaffold for IscS-mediated assembly of iron-sulfur ([Fe-S]) clusters. We report the NMR solution structure of monomeric Haemophilus influenzae IscU with zinc bound at the [Fe-S] cluster assembly site. The compact core of the globular structure has an alpha-beta sandwich architecture with a three-stranded antiparallel beta-sheet and four alpha-helices. A nascent helix is located N-terminal to the core structure. The zinc is ligated by three cysteine residues and one histidine residue that are located in and near conformationally dynamic loops at one end of the IscU structure. Removal of the zinc metal by chelation results in widespread loss of structure in the apo form. The zinc-bound IscU may be a good model for iron-loaded IscU and may demonstrate structural features found in the [Fe-S] cluster bound form. Structural and functional similarities, genomic context in operons containing other homologous genes, and distributions of conserved surface residues support the hypothesis that IscU protein domains are homologous (i.e. derived from a common ancestor) with the SufE/YgdK family of [Fe-S] cluster assembly proteins.
Journal of Molecular Biology 12/2004; 344(2):567-83. · 4.00 Impact Factor
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Journal of Biomolecular NMR 09/2004; 29(4):549-50. · 3.61 Impact Factor
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Journal of Biomolecular NMR 07/2004; 29(4):549-550. · 3.61 Impact Factor
