Solution NMR structure of the ribosomal protein RP-L35Ae from Pyrococcus furiosus.

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.

A large conformational change in the putative ATP pyrophosphatase PF0828 induced by ATP binding.

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.

Preparation of protein samples for NMR structure, function, and small-molecule screening studies.

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.

The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium.

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.

Engineering of a wheat germ expression system to provide compatibility with a high throughput pET-based cloning platform.

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.

Protein production and purification.

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.

Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium.

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.

Solution NMR structure of the iron-sulfur cluster assembly protein U (IscU) with zinc bound at the active site.

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.

The SufE sulfur-acceptor protein contains a conserved core structure that mediates interdomain interactions in a variety of redox protein complexes.

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.