Information management for proteomics: a perspective.

Proteomics is a data-rich discipline that makes extensive use of separation tools, mass spectrometry and bioinformatics to analyze and interpret the features and dynamics of the proteome. A major challenge for the field is how proteomics data can be stored and managed, such that data become permanent and can be mined with current and future tools. This article details our experience in the development of a commercial proteomic information management system. We identify the challenges faced in data acquisition, workflow management, data permanence, security, data interpretation and analysis, as well as the solutions implemented to address these issues. We finally provide a perspective on data management in proteomics and the implications for academic and industry-based researchers working in this field.

A brain dead donor model of porcine orthotopic cardiac transplantation for assessment of cardiac allograft preservation.

BACKGROUND: We aimed to develop a large animal model of orthotopic cardiac transplantation, incorporating donor brain death, to assess new methods of preservation of the donor heart. METHODS: Brain death was achieved in the donor pig by inflation of a 20 cc subdural balloon 1 h prior to harvest. The donor heart was stored for 6 h with conventional hypothermic ischaemic preservation. It was then transplanted orthotopically into the recipient pig using the Lower and Shumway technique. One hour after reperfusion, the transplanted heart was weaned from cardiopulmonary bypass with dobutamine support. Dobutamine support was continued for up to 4 h, if required. After 6 h of physiological and biochemical evaluation, the recipient was euthanased and the heart excised for histological assessment. RESULTS: All pigs experienced the classical haemodynamic changes associated with brain death. This resulted in the release of Troponin I, consistent with myocardial injury. The donor operation was successfully completed in 11 out of 13 pigs. Six out of 11 transplanted hearts were successfully weaned from cardiopulmonary bypass, but required ongoing dobutamine support. CONCLUSIONS: This porcine model of orthotopic cardiac transplantation is a relevant and practical large animal model for the assessment of new methods of preservation of the donor heart.

High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer.

The solution structure of the c-Jun leucine zipper domain has been determined to high resolution using a new calculation protocol designed to handle highly ambiguous sets of interproton distance restraints. The domain comprises a coiled coil of parallel alpha-helices in which most of the hydrophobic residues are buried at the highly symmetrical dimer interface; this interface extends over 10 helical turns and is the most elongated protein domain solved to date using NMR methods. The backbone fold is very similar to that seen in crystal structures of the GCN4 and Jun-Fos leucine zippers; however, in contrast with these crystal structures, the Jun leucine zipper dimer appears to be devoid of favorable intermolecular electrostatic interactions. A polar asparagine residue, located at the dimer interface, forms the sole point of asymmetry in the structure; furthermore, the side chain of this residue is disordered due to motional averaging. This residue, which is highly conserved in the leucine zipper family of transcription factors, provides a destabilizing influence that is likely to facilitate the rapid exchange of zipper strands in vivo.

Nuclear magnetic resonance characterization of the Jun leucine zipper domain: unusual properties of coiled-coil interfacial polar residues.

Leucine zippers constitute a widely observed structural motif which serves to promote both homo- and heterodimerization in a number of DNA-binding proteins. As part of our ongoing efforts to characterize both the structure and the dynamical properties of this dimerization domain as they relate to biological function, we report here the secondary structure in solution of a recombinant dimeric peptide (rJunLZ) comprising residues Arg276-Asn314 of the leucine zipper domain of c-Jun. Two- and three-dimensional homo- and heteronuclear NMR experiments have allowed definition of the secondary structure of rJunLZ and have provided a total of approximately 1500 interproton distance and 62 phi dihedral angle constraints for tertiary structure calculations. Amide proton protection factors, calculated from hydrogen-deuterium exchange experiments, have identified 62 hydrogen bonds in the rJunLZ dimer. We have also examined the role of Asn22, the only polar residue situated at the hydrophobic dimer interface. Virtually all leucine zipper sequences contain such a polar residue (usually Asn) near the center of the motif. X-ray crystallographic studies showed that, in the case of the GCN4 homodimer, the polar residue (Asn) adopts an asymmetric conformation in an otherwise essentially symmetric structure. In contrast, all NMR studies of leucine zipper homodimers to date have suggested that the dimers are completely symmetric in solution. We present evidence that the side-chain amide protons of Asn22 are hydrogen-bonded in solution and that this side chain exchanges rapidly between two distinct conformations. On the basis of these observations, we propose a dynamic model which can explain the apparent differences in symmetry observed in NMR and X-ray crystallographic studies of leucine zipper homodimers. We show that mutation of Asn22 to a hydrophobic Leu residue markedly increases the thermal stability of the rJunLZ homodimer, consistent with a destabilizing role for this residue. However, at temperatures below 30 degrees C, the Asn22-->Leu mutant rearranges to form oligomers larger than the dimer, as was previously observed for the corresponding Asn-->Val mutation in the GCN4 leucine zipper. These results are consistent with the hypothesis that the polar Asn residue commonly observed at the interface of leucine zippers imposes specificity for the dimer structure at the expense of stability [Harbury, P.B., Zhang, T., Kim, P.S., & Alber, T. (1993) Science 262, 1401-1407].

Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain.

Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of c-Jun plus a total of five engineered non-native residues at the N- and C-termini. The junLZ gene was cloned into the pGEX-2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (approximately 15% total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carboxy-terminal portion of Schistosoma japonicum glutathione S-transferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15N-labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel alpha-helices. We also present 15N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy.

Determination of the structure of symmetric coiled-coil proteins from NMR data: application of the leucine zipper proteins Jun and GCN4.

Previous attempts to determine the solution structures of homodimeric 'leucine zippers' using nuclear magnetic resonance (NMR) spectroscopy have been impeded by the complete symmetry of these coiled-coil molecules, which makes it impossible a priori to distinguish between intra- and intermonomer dipolar connectivities. Consequently, a number of ad hoc approaches have been used in an attempt to derive tertiary solution structures of these molecules from the NMR data. In this paper we present a more rigorous approach for analysing the NMR spectra of symmetric coiled-coil proteins. This analysis is based on calculations of intra- and intermonomer interproton distances in the recently determined crystal structure of the GCN4 leucine zipper [O'Shea, E.K., Klemm, J.D., Kim, P.S. and Alber, T. (1991) Science, 254, 539-543] and in symmetric coiled-coil models of the leucine zippers of GCN4 and the human oncoprotein Jun which we constructed using a dynamic simulated annealing approach. This analysis has enabled the formulation of a set of rules for interpreting the NMR spectra of symmetric coiled-coil proteins and has also led to the prediction of novel dipolar connectivities which we demonstrate in a 2-D NMR spectrum of the homodimeric Jun leucine zipper.

The solution structure of the leucine zipper motif of the Jun oncoprotein homodimer.

Proton NMR studies have been performed on a 9.8-kDa synthetic fragment comprising the homodimeric leucine zipper domain of the human oncoprotein Jun to ascertain its conformation in aqueous solution. Analysis of two-dimensional scalar and dipolar-coupling experiments enabled almost all proton resonances to be sequence-specifically assigned and further revealed that the Jun leucine zipper forms a completely symmetric dimer in solution, consistent with the formation of a coiled-coil arrangement of parallel alpha-helical strands. The rates of exchange of individual amide protons with solvent, as well as hydrogen-bond lengths predicted from amide proton chemical shifts, are shown to correlate with residue position in the coiled-coil. A subset of 209 unambiguous distance constraints was compiled using rules recently formulated for interpreting the NOESY spectra of symmetric coiled-coils, and these were used in combination with experimentally determined hydrogen bond and dihedral angle constraints to compute a solution structure for the Jun leucine zipper domain.

Conformation of sarafotoxin-6b in aqueous solution determined by NMR spectroscopy and distance geometry.

The solution structure of sarafotoxin-6b in water has been determined using high-resolution NMR spectroscopy. 127 proton-proton distance measurements and three phi dihedral angle constraints derived from NMR spectra were used to calculate the solution structure using a combination of distance geometry and restrained molecular dynamics. The major structural feature of the resulting family of five structures was a right-handed alpha-helix extending from K9 to Q17. In contrast, the C-terminal region of the peptide appears not to adopt a preferred conformation in aqueous solution. The present structure is compared with those previously determined for endothelin peptides in non-aqueous solvents.

Accidental intubation of the oesophagus.

It is widely accepted and taught that the accidental placement of a tracheal tube in the oesophagus can be readily detected if it is looked for, though it is recognised that death from this cause occurs from time to time. Evidence is now presented of instances where anaesthetists have been misled by a range of tests which are commonly used to check the correct placement of a tracheal tube. An explanation is offered for this unexpected finding, and some recommendations are formulated to improve patient safety.