Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA.

The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.

Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs.

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis.

Recovery of new integron classes from environmental DNA.

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.

Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies.

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [(15)N]NH(4)Cl and [(13)C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly (13)C,(15)N-labeled protein secreted by approximately 10(10) D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional (1)H-(13)C HSQC spectrum confirms (13)C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.

The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.

Solution dynamics and secondary structure of murine leukemia inhibitory factor: a four-helix cytokine with a rigid CD loop.

Leukemia inhibitory factor (LIF) is a hematopoietic cytokine which elicits its effects on diverse cell types via both gp130 and a more specific LIF receptor. Recombinant murine LIF was studied by multidimensional homonuclear and 1H-15N heteronuclear NMR and 95% of backbone amide resonances assigned. Definition of the secondary structure by chemical shift data and NOE connectivities shows a four-alpha-helix bundle fold (helices A-D) in solution, with an additional flexible turn of helix in the AB loop. Subtle differences are seen in the conformations of helices A and D from those defined in the crystal structure [Robinson, R. C., Grey, L. M., Staunton, D., Vankelcom, H., Vernallis, A. B., Moreau, J.-F., Stuart, D. I., Heath, J. K., & Jones, E. Y. (1994) Cell77, 1101-1116]. The dynamics of the polypeptide backbone of LIF were assessed from 15N T1 and T2 relaxation times and 15N-1H heteronuclear NOEs of the amide groups. Using model-free formalism, the overall rotational correlation time of LIF in solution is calculated to be 9.7 ps. The four alpha-helices are relatively rigid, and high mobility is observed for N-terminal residues (Ser 1-Asn 21) and the AB loop. In contrast to several closely related cytokines, the long CD loop is relatively rigid. This may have implications for interactions with the specific LIF receptor, which binds in this region.

Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum.

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution.

Recombinant prespore-specific antigen from Dictyostelium discoideum is a beta-sheet glycoprotein with a spacer peptide modified by O-linked N-acetylglucosamine.

Prespore-specific antigen (PsA) is a putative cell-adhesion molecule of the cellular slime mould Dictyostelium discoideum, which has a similar molecular architecture to several mammalian cell-surface proteins. It has an N-terminal globular domain presented to the extracellular environment on an O-glycosylated stem (glycopeptide) that is attached to the cell membrane through a glycosyl-PtdIns anchor. The sequence of PsA suggests that PsA may belong to a new family of cell-surface molecules and here we present information on the structure of the N-terminal globular domain and determine the reducing-terminal linkage of the O-glycosylation. To obtain a sufficient amount of pure protein, a secreted recombinant form of PsA (rPsA), was expressed in D. discoideum and characterised. 1H-NMR spectra of rPsA contained features consistent with a high degree of beta-sheet in the N-terminal globular domain, a feature commonly observed in cell-adhesion proteins. Solid-phase Edman degradation of the glycopeptide of rPsA indicated that 14 of the 15 threonines and serines in the spacer region were glycosylated. The chemical structures of the O-glycosylations were determined to be single N-acetylglucosamine residues.

NMR studies of the solution properties of recombinant murine interleukin-6.

The effects of solvent, pH and temperature on the 1H-NMR spectra of recombinant murine interleukin-6 (IL-6) are described. Assignments made from two-dimensional homonuclear spectra are presented for resonances of the fifteen aromatic amino-acid side chains. A time-dependent loss of intensity was observed for all resonances in the spectrum of IL-6, probably as a result of aggregation. This aggregation is markedly temperature-dependent. The pKa values of the four histidine residues in murine IL-6 has been measured; one has a value of 5.5, approx. one pH unit less than the value exhibited by the other three. Analysis of the NOESY spectra has allowed a preliminary characterisation of the nature of interactions among the aromatic side chains within the protein fold. 1H and 15N resonances of residues Thr-4 to Val-21 are assigned from three-dimensional 1H-15N correlated spectroscopy, and evidence is presented for these residues comprising a mobile N-terminal tail with little ordered structure. An N-terminal mutant lacking the first 22 residues of the murine IL-6 sequence and known to possess full biological activity was also examined and shown to have essentially retained the tertiary fold of the native molecule.

Linear and cyclic peptide analogues of the polypeptide cardiac stimulant, anthopleurin-A. 1H-NMR and biological activity studies.

A loop corresponding to residues 8-17 in the polypeptide cardiac stimulant anthopleurin-A is known to be important for the cardiostimulant activity of this molecule. To investigate the activity and possible conformations of this loop in isolation, two synthetic peptides have been studied. The first corresponds to residues 6-20 of anthopleurin-A with Cys6 replaced by Thr, and the second to residues 6-21 of anthopleurin-A, with Thr21 replaced by Cys. The introduction of an additional cysteine in the latter peptide enabled an intramolecular disulfide to be formed between the N- and C-terminal residues. Both linear peptides and the disulfide-containing analogue lack the cardiostimulant and Na(+-)-channel binding activity in the parent molecule, anthopleurin-A, indicating that although the loop is important for the function of anthopleurin-A, other regions of the molecule must also be involved in activity. Assignments of the 1H-NMR spectra of both peptides are presented, and their pH and temperature dependences investigated. The results show that the amide protons of Gly5 and Asn11 (corresponding to Gly10 and Asn16 in anthopleurin-A) sample hydrogen-bonded conformations in solution. Based on these NMR data, two regions of non-random structure, encompassing residues 2-5 and 8-11, respectively, are proposed, and the possible involvement of such structures in the activity of anthopleurin-A is discussed.

Structure-function relationships in the polypeptide cardiac stimulant, anthopleurin-A. Effects of limited proteolysis by trypsin.

Selective proteolysis of the polypeptide cardiostimulant anthopleurin-A by trypsin introduces a single break in the polypeptide backbone on the C-terminal side of Arg14. The resulting derivative is devoid of any cardiostimulant activity. The structural changes which accompany this loss of activity have been examined by one- and two-dimensional 1H-NMR spectroscopy. It is shown that the overall backbone folding of anthopleurin-A is conserved on digestion, with some structural changes occurring for residues which are adjacent to the site of cleavage by trypsin. Thus, although previous NMR studies on anthopleurin-A indicate that the region surrounding Arg14 is devoid of any ordered structure, it appears that some degree of structural integrity is required to allow the essential side chains to adopt the conformation necessary to produce a cardiostimulant effect.

Sequential 1H-NMR assignments and secondary structure of the sea anemone polypeptide anthopleurin-A.

The sequence-specific assignment of resonances in the 500-MHz 1H-NMR spectrum of a cardioactive sea anemone polypeptide, anthopleurin-A, is described. The assignment procedure involved analysis of two-dimensional phase-sensitive multiple-quantum-filtered, double-quantum, homonuclear Hartmann-Hahn and nuclear Overhauser effect spectra. Using sequential information, specific assignments have been made for resonances arising from all 49 amino acid residues. Resonances arising from a number of residues in a minor conformer present in solution are also assigned. These results greatly extend previous resonance assignments made from spectra acquired at 300 MHz [Gooley, P. R. and Norton, R. S. (1985) Eur. J. Biochem. 153, 529-539] and provide the basis for a more accurate definition of the conformation of anthopleurin-A in aqueous solution. The secondary structure includes a four-stranded antiparallel beta-sheet encompassing residues 2-4, 21-23, 34-36 and 45-49, and possibly a beta-bulge located at Ser-19 and Gly-20. A type II beta-turn is formed by residues 30-33. These structural elements also occur within other related sea anemone polypeptides, but the conformation of the small loop region containing Pro-41 appears to be unique to anthopleurin-A.

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I.

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.

Backbone folding of the polypeptide cardiac stimulant anthopleurin-A determined by nuclear magnetic resonance, distance geometry and molecular dynamics.

The solution conformation of the cardiac stimulatory sea anemone polypeptide anthopleurin-A has been characterised using distance geometry and restrained molecular dynamics calculations. A set of 253 approximate interproton distance restraints and 14 peptide backbone torsion angle restraints derived from two-dimensional 1H-NMR spectra at 500 MHz were used as input for these calculations. 13 structures generated by either metric matrix or variable target function distance geometry calculations were refined using energy minimisation and restrained molecular dynamics. The resulting structures contain a region of twisted antiparellel beta-sheet to which two separate regions of unordered chain are linked by three disulphide bonds. Two loops, one including Pro-41 and the other encompassing residues 10-18, are poorly defined by the NOE data.

Two-dimensional 1H-NMR studies of the solution structure of plasminogen kringle 4.

Native kringle 4 from human plasminogen has been studied by two-dimensional 1H-NMR methods in order to obtain new structural information about the kringle fold. Two-dimensional scalar correlated spectroscopy (COSY), two-dimensional dipolar correlated spectroscopy (NOESY) and two-dimensional relayed coherance transfer spectroscopy (RCT) experiments were recorded, allowing most resonances arising from the aromatic and methyl-containing residues to be assigned in the spectrum. From an analysis of NOE data, a small segment of double-stranded beta-sheet has been identified near residues Phe63 and Thr64. Further analysis of the NOESY spectrum has allowed detailed study of the conformation of sidechains located in regions near Leu45 and Val69. A model has been constructed of the polypeptide segment comprising residues 40-49 which accounts for the observed NOE interactions.

Assignment of heme and distal amino acid resonances in the 1H-NMR spectra of the carbon monoxide and oxygen complexes of sperm whale myoglobin.

Assignments of resonances of the heme and distal amino acid protons in spectra of the CO and O2 complexes of sperm whale myoglobin are reported. These resonances provide information on the conformation of the heme pocket. For oxymyoglobin, the assignments of the heme meso protons disagree with those proposed previously on the basis of partial deuteration experiments. Rapid ring flips about the C beta-C gamma bond are detected for Phe-CD1. Recent claims for two conformational substates of valine-E11 in carbonmonoxymyoglobin (Bradbury, J.H. and Carver, J.A. (1984) Biochemistry 23, 4905-4913) are shown to be in error. The pK of His-97 (FG3) in carbonmonoxymyoglobin has been determined (pK = 5.9). This residue appears to influence many spectroscopic properties of myoglobin. The distal His-E7 in carbonmonoxymyoglobin has pK less than 5.0. Differences in the heme pocket conformation in the CO complexes of myoglobin and leghemoglobin are discussed. These differences may be influential in O2 and CO association reactions.

NMR studies of oxyleghemoglobin: assignment of distal histidine proton resonances and evidence for pH-dependent changes in conformation.

The resonance of the C-2 proton of the distal histidine has been assigned in the 400 MHz 1H-NMR spectrum of soybean ozyleghemoglobin a. This resonance is subject to a very large ring current shift from the heme and occurs to high field of the residual HO2H peak. The pH dependence was measured from a series of nuclear Overhauser effect difference spectra over a range of pH values. The resonance moves to high field with decreasing pH and reflects titration of a one proton-dissociable group with pK 5.5. Resonances of the heme substituents and distal amino acid side-chains are also sensitive to this titration. Changes in ring-current shifts and nuclear Overhauser effects indicate that a conformational change occurs in the heme pocket upon titration of the pK 5.5 group. We propose that protonation of the distal histidine with pK 5.5 is accompanied by movement of the imidazole ring towards the heme normal. This movement would allow interaction between the ligated oxygen molecule and the protonated distal histidine at acid pH.