Phillip S. Brereton

Backbone solution structures of proteins using residual dipolar couplings: application to a novel structural genomics target

Structural genomics (or proteomics) activities are critically dependent on the availability of high-throughput structure determination methodology. Development of such methodology has been a particular challenge for NMR based structure determination because of the demands for isotopic labeling of proteins and the requirements for very long data acquisition times. We present here a methodology that gains efficiency from a focus on determination of backbone structures of proteins as opposed to full structures with all sidechains in place. This focus is appropriate given the presumption that many protein structures in the future will be built using computational methods that start from representative fold family structures and replace as many as 70% of the sidechains in the course of structure determination. The methodology we present is based primarily on residual dipolar couplings (RDCs), readily accessible NMR observables that constrain the orientation of backbone fragments irrespective of separation in space. A new software tool is described for the assembly of backbone fragments under RDC constraints and an application to a structural genomics target is presented. The target is an 8.7 kDa protein from Pyrococcus furiosus, PF1061, that was previously not well annotated, and had a nearest structurally characterized neighbor with only 33% sequence identity. The structure produced shows structural similarity to this sequence homologue, but also shows similarity to other proteins, which suggests a functional role in sulfur transfer. Given the backbone structure and a possible functional link this should be an ideal target for development of modeling methods.

The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k(cat)/K(m) value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108 degrees C that increases to 112 degrees C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-A resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.

A pure S = 3/2 [Fe4S4]+ cluster in the A33Y variant of Pyrococcus furiosus ferredoxin.

The properties of the [4Fe‐4S]2+/+ cluster in wild‐type and the A33Y variant of Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR, variable‐temperature magnetic circular dichroism (VTMCD) and resonance Raman (RR) spectroscopies. The A33Y variant involves the replacement of an alanine whose α‐C is less than 4 Å from one of the cluster iron atoms by a tyrosine residue. Although the spectroscopic results give no indication of tyrosyl cluster ligation, the presence of a tyrosine residue in close proximity to the cluster results in a 38‐mV decrease in the midpoint potential of the [4Fe‐4S]2+,+ couple and has a marked effect on the ground state properties of the reduced cluster. The mixed spin [4Fe‐4S]+ cluster in the wild‐type protein, 80% S=3/2 (E/D=0.22, D=+3.3 cm−1) and 20% S=1/2 (g=2.10, 1.87, 1.80), is converted into a homogeneous S=3/2 (E/D=0.30, D=−0.7 cm−1) form in the A33Y variant. As the first example of a pure S=3/2 [4Fe‐4S]+ cluster in a ferredoxin, this variant affords the opportunity for detailed characterization of the excited electronic properties via VTMCD studies and demonstrates that the protein environment can play a crucial role in determining the ground state properties of [4Fe‐4S]+ clusters.

Structure determination of a new protein from backbone-centered NMR data and NMR-assisted structure prediction

Targeting of proteins for structure determination in structural genomic programs often includes the use of threading and fold recognition methods to exclude proteins belonging to well‐populated fold families, but such methods can still fail to recognize preexisting folds. The authors illustrate here a method in which limited amounts of structural data are used to improve an initial homology search and the data are subsequently used to produce a structure by data‐constrained refinement of an identified structural template. The data used are primarily NMR‐based residual dipolar couplings, but they also include additional chemical shift and backbone‐nuclear Overhauser effect data. Using this methodology, a backbone structure was efficiently produced for a 10 kDa protein (PF1455) from Pyrococcus furiosus. Its relationship to existing structures and its probable function are discussed. Proteins 2006.

High-throughput production of Pyrococcus furiosus proteins: considerations for metalloproteins.

Free-living prokaryotic organisms contain all of the proteins required for the basic biochemical processes of life. As part of the Southeastern Collaboratory for Structural Genomics (SECSG), Pyrococcus furiosus is being used as a model system for developing a high-throughput protein expression and purification protocol. Its 1.9 million basepair genome encodes approximately 2200 putative proteins, less than 25% of which show similarity to any structurally characterized protein in the Protein Data Bank. The overall goal of the structural genomics initiative is to determine, in total, all existing protein folds. The immediate objective of this work is to obtain recombinant forms of all P. furiosus proteins in their functional states for structural determination. Proteins successfully produced by overexpression in another organism such as the bacterium Escherichia coli typically contain a single subunit, are soluble and do not contain (complex) cofactors. Analyses of the P. furiosus genome suggest that perhaps only a quarter of the genes encode proteins that would fall into this category. The hypothesis is that lack of the appropriate cofactor or of the partner protein(s) necessary to form a complex are major reasons why many recombinant proteins are insoluble. This work describes development of the production pipeline with attention to prediction and incorporation of cofactors.

[3] Ferrodoxin from Pyrococcus furiosus

Publisher Summary Ferredoxins (Fd) are small proteins that contain iron-sulfur clusters as a redox active group. They are ubiquitous in biological systems and play integral roles in a wide variety of electron transfer processes, including respiration, photosynthesis, and fermentation. There are two main varieties: the 4Fe-type, which contain one and sometimes two cubane-type [4Fe-4S] clusters, and the 2Fe-type, which contain a [2Fe-2S] cluster (the S represents inorganic sulfide). Both types are covalently attached to their proteins via Fe-S bonds between the Fe atoms of the cluster and the sulfur atoms of four cysteine residues. Ferredoxins have been purified from a variety of microbial and eukaryotic sources and have been extensively studied. The first to be characterized from a hyperthermophile was from Pyrococcus furiosus ; in fact, this was one of the first proteins to be obtained from such an organism. The ease of purification and remarkable stability of P. furiosus ferredoxin, together with the availability of the recombinant protein, has enabled it to become one of the best studied of all ferredoxins, with numerous investigations into the properties of both the protein and of its single [4Fe-4S] cluster. This chapter describes the purification of ferredoxin from P. furiosus , and the purification of the recombinant protein from Escherichia coli , together with several mutant forms. Some of the methods that have been developed in characterizing this protein are also described in the chapter.