Zsolt Zolnai

Project management system for structural and functional proteomics: Sesame

A computing infrastructure (Sesame) has been designed to manage and link individual steps in complex projects. Sesame is being developed to support a large-scale structural proteomics pilot project. When complete, the system is expected to manage all steps from target selection to data-bank deposition and report writing. We report here on the design criteria of the Sesame system and on results demonstrating successful achievement of the basic goals of its architecture. The Sesame software package, which follows the client/server paradigm, consists of a framework, which supports secure interactions among the three tiers of the system (the client, server, and database tiers), and application modules that carry out specific tasks. The framework utilizes industry standards. The client tier is written in Java2 and can be accessed anywhere through the Internet. All the development on the server tier is also carried out in Java2 so as to accommodate a wide variety of computer platforms. The database tier employs a commercial database management system. Each Sesame application module consists of a simple user interface in the client tier, corresponding objects in the server tier, and relevant data stored in the centralized database. For security, access to stored data is controlled by access privileges. The system facilitates both local and remote collaborations. Because users interact with the system using Java Web Start or through a web browser, access is limited only by the availability of an Internet connection. We describe several Sesame modules that have been developed to the point where they are being utilized routinely to support steps involved in structural and functional proteomics. This software is available to parties interested in using it and assisting to guide its further development.

New bioinformatics resources for metabolomics.

We recently developed two databases and a laboratory information system as resources for the metabolomics community. These tools are freely available and are intended to ease data analysis in both MS and NMR based metabolomics studies. The first database is a metabolomics extension to the BioMagResBank (BMRB, http://www.bmrb.wisc.edu), which currently contains experimental spectral data on over 270 pure compounds. Each small molecule entry consists of five or six one- and two-dimensional NMR data sets, along with information about the source of the compound, solution conditions, data collection protocol and the NMR pulse sequences. Users have free access to peak lists, spectra, and original time-domain data. The BMRB database can be queried by name, monoisotopic mass and chemical shift. We are currently developing a deposition tool that will enable people in the community to add their own data to this resource. Our second database, the Madison Metabolomics Consortium Database (MMCD, available from http://mmcd.nmrfam.wisc.edu/), is a hub for information on over 10,000 metabolites. These data were collected from a variety of sites with an emphasis on metabolites found in Arabidopsis. The MMC database supports extensive search functions and allows users to make bulk queries using experimental MS and/or NMR data. In addition to these databases, we have developed a new module for the Sesame laboratory information management system (http://www.sesame.wisc.edu) that captures all of the experimental protocols, background information, and experimental data associated with metabolomics samples. Sesame was designed to help coordinate research efforts in laboratories with high sample throughput and multiple investigators and to track all of the actions that have taken place in a particular study.

Analysis of laboratory-frame and rotating-frame cross-relaxation buildup rates from macromolecules

Abstract A quadratic approximation of cross-relaxation buildup rates is described for identifying and correcting for indirect magnetization transfer effects in macromolecular spectra. This approach has permitted the accurate determination of interproton distances in a protein of molecular weight 6000, turkey ovomucoid third domain. An illustration is given of the analysis of a three-spin system to yield a distance between two protons that lacked resolved direct NOESY and ROESY cross peaks.

Solution structures of staphylococcal nuclease from multidimensional, multinuclear NMR: nuclease-H124L and its ternary complex with Ca2+ and thymidine-3',5'-bisphosphate.

The solution structures of staphylococcal nuclease (nuclease) H124L and itsternary complex, (nuclease-H124L)•pdTp•Ca2+, were determinedby ab initio dynamic simulated annealing using 1925 NOE, 119 φ, 20χ1 and 112 hydrogen bond constraints for the free protein,and 2003 NOE, 118 φ, 20 χ1 and 114 hydrogen bondconstraints for the ternary complex. In both cases, the final structuresdisplay only small deviations from idealized covalent geometry. In structuredregions, the overall root-mean-square deviations from mean atomic coordinatesare 0.46 (±0.05) Å and 0.41 (±0.05) Å for thebackbone heavy atoms of nuclease and its ternary complex, respectively. Thebackbone conformations of residues in the loop formed byArg81–Gly86, which is adjacent to the activesite, are more precisely defined in the ternary complex than in unligatednuclease. Also, the protein side chains that show NOEs and evidence forhydrogen bonds to pdTp (Arg35, Lys84,Tyr85, Arg87, Tyr113, andTyr115) are better defined in the ternary complex. As has beenobserved previously in the X-ray structures of nuclease-WT, the binding ofpdTp causes the backbone of Tyr113 to change from an extendedto a left-handed α-helical conformation. The NMR structures reportedhere were compared with available X-ray structures: nuclease-H124L [Truckseset al. (1996) Protein Sci., 5, 1907–1916] and the ternary complex ofwild-type staphylococcal nuclease [Loll and Lattman (1989) Proteins Struct.Funct. Genet., 5, 183–201]. Overall, the solution structures ofnuclease-H124L are consistent with these crystal structures, but smalldifferences were observed between the structures in the solution and crystalenvironments. These included differences in the conformations of certain sidechains, a reduction in the extent of helix 1 in solution, and many fewerhydrogen bonds involving side chains in solution.

The Center for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) is a “specialized” or “technology development” center supported by the Protein Structure Initiative (PSI). CESG’s mission is to develop improved methods for the high-throughput solution of structures from eukaryotic proteins, with a very strong weighting toward human proteins of biomedical relevance. During the first three years of PSI-2, CESG selected targets representing 601 proteins from Homo sapiens, 33 from mouse, 10 from rat, 139 from Galdieria sulphuraria, 35 from Arabidopsis thaliana, 96 from Cyanidioschyzon merolae, 80 from Plasmodium falciparum, 24 from yeast, and about 25 from other eukaryotes. Notably, 30% of all structures of human proteins solved by the PSI Centers were determined at CESG. Whereas eukaryotic proteins generally are considered to be much more challenging targets than prokaryotic proteins, the technology now in place at CESG yields success rates that are comparable to those of the large production centers that work primarily on prokaryotic proteins. We describe here the technological innovations that underlie CESG’s platforms for bioinformatics and laboratory information management, target selection, protein production, and structure determination by X-ray crystallography or NMR spectroscopy.

Quantitative evaluation of two-dimensional cross-relaxation NMR spectra of proteins. Interproton distances in Turkey ovomucoid third domain

Abstract A novel method is proposed for quantifying two-dimensional, phase-sensitive, cross-relaxation spectra of proteins. Relative cross-peak volumes are calculated from peak heights and linewidths (measured along the x and y axes). We show that this method gives the same result for isolated peaks as direct volume integration. In the case of moderate peak overlap, our method is less prone to error than volume integration. Computerization of the method is easily implemented and can be used for measuring the massive sets of cross-peak volumes contained in a series of two-dimensional NMR spectra. This approach has enabled us to use a quadratic approximation to the initial build-up rates to determine cross-relaxation rates for 90 proton pairs in a protein, in the laboratory and rotating frames of reference (J. Fejzo, Zs. Zolnai, S. Macura, and J. L. Markley, J. Magn. Reson. 82, 518, 1989). The molecule studied was turkey ovomucoid third domain (OMTKY3), a small (6.1 kDa) globular protein. The cross-relaxation rates in the two frames were analyzed in terms of molecular mobility and interproton distances. The results confirmed that rigid-body isotropic motion with a correlation time of 6 ns holds for OMTKY3. Interproton distances were determined with relative errors of 10–30% and compared with values derived from X-ray crystal structure of this protein.

Magnetization exchange network editing: mathematical principles and experimental demonstration

Abstract We have examined analytically the effects of different NMR magnetization exchange network editing (MENE) procedures, starting from equilibrium or nonequilibrium conditions, on generalized systems that undergo magnetization exchange (cross-relaxation and/or chemical exchange). Larger errors in derived exchange rate constants are found in experiments that start from nonequilibrium states than in those that start from equilibrium. On the other hand, editing experiments that decompose the dynamic matrix into two or more block diagonal submatrices generally yield more accurate exchange rate constants. Theoretical arguments, backed up by experimental results, demonstrate that MENE experiments lead to increased rates of relaxation for the edited cross and diagonal peaks. We describe a new class of heteronuclear editing experiment in which the system starts from a nonequilibrium state and is edited during the mixing period. In an experimental application of this approach to the spectral analysis of a small protein, recombinant human ubiquitin (M r 8565) labeled uniformly with 15 N and 13 C, we demonstrate how the cross-relaxation network of the protein can be decomposed simultaneously into subnetworks of 15 N-bound protons, aliphatic 13 C-bound protons, aromatic 13 C-bound protons, and ( 12 C/ 14 N/O)-bound protons. Such a decomposition permits the measurement of slower magnetization exchange rates, including those that are masked in conventional cross-relaxation experiments.

Compression of NMR data. Application to two-dimensional NMR spectroscopy and imaging

Abstract The important information in a high-resolution two-dimensional NMR spectrum or 2D NMR image is localized in a small fraction of the overall data block. Hence data files should be subject to compression by suitable manipulation. We propose two methods for data compression: elimination of background noise and logarithmic scaling of the data. By combining the two methods, one can obtain a compression factor of 30 or more without significant loss of spectral information content. The method is particularly suitable for the storage of massive data sets. One or more two-dimensional spectra can be reduced and stored on one floppy disk. These methods and their versatility are demonstrated via compression of two-dimensional NMR spectra of a small protein (turkey ovomucoid third domain) and via compression of the NMR image of a phantom.

Drafting table and light-box software for multidimensional NMR spectral analysis (PIXI). The personal computer workstation☆

Abstract We have developed a fast, multidimensional NMR spectral analysis program (PIXI) that runs on personal computers built around the Intel 80X86 family of microprocessors with EGA-compatible video graphics controllers. The program operates on compressed data sets that can be extracted from spectra processed by any NMR processing software package. Up to eight different spectra can be loaded simultaneously, and each spectrum can be expanded into nine regions. All of the eight full spectra and the 72 expanded regions are accessible for rapid display with three or fewer keystrokes. Features of PIXI include contour plots with up to 16 levels for all expansions, overlaying of up to four spectra or expansions, peak picking with storage to a disk file, and the simultaneous display of symmetric regions of spectra. The appendixes include instructions for preparing the compressed data sets required by PIXI and a wiring diagram for an adapter used in parallel transfer of data between Bruker Aspect computers and a commercially available parallel port for Intel 80X86 family computers.

Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies

Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or 1H–15N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.