Protein folding handbook, 5-volume set

ISBN 13: 9783527307845

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Unlike the class 1, most other integron classes are located in the bacterial chromosome, and have gene cassette arrays which characteristically differ to the class 1 integron arrays. The arrays can be very large, in the case of Vibrios comprising hundreds of cassettes [10] , [11].

The potential impact of the integron in shaping bacterial evolution is largely dependent on the extent to which the mobilised gene cassettes replicate functions already resident within their host genome or contribute additional functions which, while not encoding essential proteins, may provide adaptive traits to the host under certain environmental conditions [14] , [15].

Most analyses of the cassette gene pool to date have been focused on sequence-based annotation. Given the high degree of novelty, these approaches are unable to enlighten as to whether the recovered genes encode additional representatives of known protein families, or in fact comprise an additional substantial reservoir of unique functions that heightens the value of an integron array in exploring new ecological niches.

One route to discerning between these two options is through the elucidation of the three-dimensional structure of the encoded protein products, which remains strongly conserved, unlike amino acid sequence [16]. If novel cassettes primarily encode sequence-divergent variants of known proteins, this can be verified through shared fold and geometry of active site; however if the overwhelming novelty of the integron is derived from the presence of many new protein families that we have not seen before, then they are likely to possess novel folds as well.

We have chosen to examine protein structures encoded by the cassette metagenome to discern the degree to which the novel gene sequences truly represent proteins of new fold and function. We have focused on integron gene cassettes recovered by cassette-PCR [17] from uncultured bacteria in environmental samples, as well as from strain isolates of Vibrio cholerae and the related Vibrio metecus formerly paracholerae. Here, we describe six cassette-encoded proteins within our final group of 19 crystal structures, each found to display a novel fold, and indicating the cassette metagenome to be remarkably rich in new protein families.

This group of structures directly accessed from integron arrays provides additional diversity to those genetic elements known to have undergone successful integron-mediated lateral transfer. Permits were not required for sampling. Small sediment samples 0. Gene cassettes from V. Strains of V. Several water samples 1 ml were spread directly on thiosulfate citrate bile salts TCBS agar selective for V.

Protein folding handbook, 5-volume set

Isolated colonies of a yellow colour sucrose positive [22] were picked and re-streaked on tryptic soy broth media. After another overnight incubation, isolated colonies were picked and re-streaked on TCBS media and again incubated overnight. SeMet-derivatised protein was produced in 1 l cultures via auto-induction using PASM media [25]. Purified protein was dialysed into 50 mM Tris buffer pH 9.

INTRODUCTION

The PHENIX suite [32] was used to solve phases from Se-derivatised methionines within each protein chain and for automated building and refinement. Manual model-building of protein chains, water molecules and bound components was performed with Coot [33]. Statistics for solution and refinement of the structures are presented in Table 1. Structures in this work display 1. Sequence homology searches were performed using Blast [40] and TBlastN [40] against the non-redundant and environmental non-redundant databases.

Analysis of subunit interactions, surface clefts, and detection of matches to active site, ligand-binding and DNA-binding templates August utilised PISA version 1. ProtParam [45] formulae were utilised to calculate M r values.

1. Introduction

Molecular images were generated with Pymol [46]. Oligomeric states were determined for some recombinant protein products by size exclusion chromatography performed at 0.

Elution volumes were calibrated with size standards The gene cassettes described in this study arise both from chromosomal integron arrays of multiple Vibrio strains V. Amino acid sequences of these structural targets are depicted in Figure 1. Sequences do not include additional affinity tags used for recombinant production.

Schematic diagrams show structures of monomer forms for proteins A. Ribbon depiction with colour spectrum from N-terminus blue to C-terminus red for each chain. Dimer interface engages hydrophobic residues from Chain A tan and Chain B green. Acidic groups located either side of a small hydrophobic pocket on the external face are indicated red.

Electrostatic surface potential of the dimer surface. Key acidic features Glu47, Asp60 and helix 2 side chains are labelled. The externally exposed face of each protomer is, by contrast, markedly acidic. Residues 60—66 of the loop appear to be most flexible, possibly modulating access for any interacting ligand at this position. The protein encoded within this single gene cassette thus presents a prominent binding groove, potentially gated by acidic residues.

No structural or sequence-based homologues are currently identifiable for this novel variant of helical fold. A loop of weak density connecting helices 2 and 3 is represented by dotted line. These same helix and loop components also contribute to hydrogen bonding and salt bridge stabilisation of the dimer. A spread of basic side groups is a distinctive feature of the exposed surface of the dimer, incorporating Arg62, Lys, Lys and Arg sidechains from both chains Figure 3.

Although there is no relationship at the sequence level, the majority of structures defined across this clan consistently show dimeric organisation mediated largely through hydrophobic residues of helix 2. Significantly, the packing geometry of these various dimeric structures are markedly different. This preserved feature likely contributes to the biochemistry of a substrate site for this protein family. Dimensions highlight distinctive flattened form. Electrostatic surface potential of the trimer surface highlights polar cavities arrowed and exposed acidic clusters on external loops.

Solvent molecules trapped within the crevice are shown spheres.

Surrounded by pronounced acidic clusters, largely from side chains on loop features, this region has the appearance of a functional binding site. A search against a database of cognate binding sites [43] , identified some features at this location common to enzymes utilising nucleotide-based cofactors e. The cytosolic zinc-binding domain of CzrB, an integral membrane transporter, aligns 2.

In CzrB, the domain presents a cluster of zinc-binding residues for metal chelation and controls a dimerisation event critical to function [50]. The 2.

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As shown in Figure 5 , this structure forms half of a structurally asymmetric tetramer. Ribbon depiction of two domain-swapped chains, each coloured from N-terminus blue to C-terminus red. Tetrameric organisation depicted in ribbon form left panel , showing engagement between two dimers green and blue , each comprising two chains. Side chain stacking of key contacting residues Tyr28, Tyr30, Arg31 and Glu35 is depicted.

Corresponding view of electrostatic surface is also shown right , with deep cleft centrally positioned.

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Rotated view of electrostatic surface, positioned to emphasise narrow dimensions of this basic slot along one tetramer surface. At the centre of the crystallised tetramer, the 3 10 helices at the edges of two opposing subunits come into contact via well-ordered stacking of protruding polar and charged side chains Tyr28, Tyr30, Arg31, Glu35; see Fig.

This contact means that the 3 10 helices from the remaining two domains are separated further out along the tetrameric interface. The flattened nature of the tetramer and the asymmetrical interactions of its component dimers results in two large faces with markedly different surface features. On one face, a narrow slot Figure 5C is formed by relatively close juxtaposition of the beta sheets from the two domains linked via contacting 3 10 helices. This cleft is lined with basic features side chains Arg16, His46 and Arg On the opposite face, the equivalent slot is very wide, due to the diagonal separation of the sheet components about the 3 10 helix interface.

Preserved residues include the key charged and aromatic groups mediating tetrameric organisation: Tyr30, Arg31, Glu32, Glu35, Arg51, Glu This linker segment also contains Arg sidechains conserving the basic chemistry of surface clefts on the two faces of the tetramer.

Protein Folding Handbook

The two opposing sheet features are thus separated across the elongated dimer, both presenting exposed faces to solvent. This helix contains a significant number of aromatic side chains Phe, Trp, Tyr, Phe which contribute hydrophobic and hydrogen-bonding stability to the dimeric interface. Located in a single asymmetric unit are dimer 1 yellow, green chains and dimer 2 pink, cyan chains. Such crystal interactions may be indicative of interactions possible for heterogenous protein partners relevant to the biological role of the protein.

Some topological relationship is detected to the Ivy virulence factor proteins e. Sequence alignment shows strongest conservation of structural residues e. Thus, should this cleft form a functional binding site for the protein, its features would be unique to the biochemistry of the Vibrio gene cassette sequence alone. Ribbon depiction of dimer coloured in spectrum from N-terminus blue to C-terminus red for each chain. Side chains contributing to the hydrophobic pocket are depicted.

The 1. Residues lining this hydrophobic binding pocket are detailed right panel. The single water molecule red sphere and unidentifiable ligand grey are also shown. The hydrogen-bonding network engaging Arg21, Tyr14, acetate and water is dashed in red. In the crystal form we have isolated, electron density consistent with a linear organic molecule is observed in this site, as shown in Figure 7.

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