Kurt Wüthrich

MOLMOL: a program for display and analysis of macromolecular structures.

MOLMOL is a molecular graphics program for display, analysis, and manipulation of three-dimensional structures of biological macromolecules, with special emphasis on nuclear magnetic resonance (NMR) solution structures of proteins and nucleic acids. MOLMOL has a graphical user interface with menus, dialog boxes, and on-line help. The display possibilities include conventional presentation, as well as novel schematic drawings, with the option of combining different presentations in one view of a molecule. Covalent molecular structures can be modified by addition or removal of individual atoms and bonds, and three-dimensional structures can be manipulated by interactive rotation about individual bonds. Special efforts were made to allow for appropriate display and analysis of the sets of typically 20-40 conformers that are conventionally used to represent the result of an NMR structure determination, using functions for superimposing sets of conformers, calculation of root mean square distance (RMSD) values, identification of hydrogen bonds, checking and displaying violations of NMR constraints, and identification and listing of short distances between pairs of hydrogen atoms.

Improved spectral resolution in COSY 1H NMR spectra of proteins via double quantum filtering

A double quantum filter is inserted into a two-dimensional correlated (COSY) 1H NMR experiment to obtain phase-sensitive spectra in which both cross peak and diagonal peak multiplets have anti-phase fine structure, and in which the cross peaks and the major contribution to the diagonal peaks have absorption lineshapes in both dimensions. The elimination of the dispersive character of the diagonal peaks in phase-sensitive, double quantum-filtered COSY spectra allows identification of cross peaks lying immediately adjacent to the diagonal, which represents a significant improvement over the conventional COSY experiment.

A TWO-DIMENSIONAL NUCLEAR OVERHAUSER ENHANCEMENT (2D NOE) EXPERIMENT FOR THE ELUCIDATION OF COMPLETE PROTON-PROTON CROSS-RELAXATION NETWORKS IN BIOLOGICAL MACROMOLECULES

Abstract The recently developed technique of two-dimensional (2D) cross-relaxation spectroscopy is utilized for systematic measurements of selective nuclear Overhauser enhancements (NOE) in the high resolution 1H nuclear magnetic resonance (NMR) spectra of biological macromolecules in solution. Compared to conventional one-dimensional NOE studies, the 2D NOE experiment has the principal advantage that it avoids detrimental effects arising from the limited selectivity of preirradiation in crowded spectral regions. Furthermore, it yields with a single instrument setting a complete network of NOEs between all the protons in the macromolecule. The resulting information on intramolecular proton-proton distances provides a new avenue for studies of the spatial structures of biopolymers.

Protein NMR Structure Determination with Automated NOE Assignment Using the New Software CANDID and the Torsion Angle Dynamics Algorithm DYANA

Combined automated NOE assignment and structure determination module (CANDID) is a new software for efficient NMR structure determination of proteins by automated assignment of the NOESY spectra. CANDID uses an iterative approach with multiple cycles of NOE cross-peak assignment and protein structure calculation using the fast DYANA torsion angle dynamics algorithm, so that the result from each CANDID cycle consists of exhaustive, possibly ambiguous NOE cross-peak assignments in all available spectra and a three-dimensional protein structure represented by a bundle of conformers. The input for the first CANDID cycle consists of the amino acid sequence, the chemical shift list from the sequence-specific resonance assignment, and listings of the cross-peak positions and volumes in one or several two, three or four-dimensional NOESY spectra. The input for the second and subsequent CANDID cycles contains the three-dimensional protein structure from the previous cycle, in addition to the complete input used for the first cycle. CANDID includes two new elements that make it robust with respect to the presence of artifacts in the input data, i.e. network-anchoring and constraint-combination, which have a key role in de novo protein structure determinations for the successful generation of the correct polypeptide fold by the first CANDID cycle. Network-anchoring makes use of the fact that any network of correct NOE cross-peak assignments forms a self-consistent set; the initial, chemical shift-based assignments for each individual NOE cross-peak are therefore weighted by the extent to which they can be embedded into the network formed by all other NOE cross-peak assignments. Constraint-combination reduces the deleterious impact of artifact NOE upper distance constraints in the input for a protein structure calculation by combining the assignments for two or several peaks into a single upper limit distance constraint, which lowers the probability that the presence of an artifact peak will influence the outcome of the structure calculation. CANDID test calculations were performed with NMR data sets of four proteins for which high-quality structures had previously been solved by interactive protocols, and they yielded comparable results to these reference structure determinations with regard to both the residual constraint violations, and the precision and accuracy of the atomic coordinates. The CANDID approach has further been validated by de novo NMR structure determinations of four additional proteins. The experience gained in these calculations shows that once nearly complete sequence-specific resonance assignments are available, the automated CANDID approach results in greatly enhanced efficiency of the NOESY spectral analysis. The fact that the correct fold is obtained in cycle 1 of a de novo structure calculation is the single most important advance achieved with CANDID, when compared with previously proposed automated NOESY assignment methods that do not use network-anchoring and constraint-combination.

The program XEASY for computer-supported NMR spectral analysis of biological macromolecules.

SummaryA new program package, XEASY, was written for interactive computer support of the analysis of NMR spectra for three-dimensional structure determination of biological macromolecules. XEASY was developed for work with 2D, 3D and 4D NMR data sets. It includes all the functions performed by the precursor program EASY, which was designed for the analysis of 2D NMR spectra, i.e., peak picking and support of sequence-specific resonance assignments, cross-peak assignments, cross-peak integration and rate constant determination for dynamic processes. Since the program utilizes the X-window system and the Motif widget set, it is portable on a wide range of UNIX workstations. The design objective was to provide maximal computer support for the analysis of spectra, while providing the user with complete control over the final resonance assignments. Technically important features of XEASY are the use and flexible visual display of ‘strips’, i.e., two-dimensional spectral regions that contain the relevant parts of 3D or 4D NMR spectra, automated sorting routines to narrow down the selection of strips that need to be interactively considered in a particular assignment step, a protocol of resonance assignments that can be used for reliable bookkeeping, independent of the assignment strategy used, and capabilities for proper treatment of spectral folding and efficient transfer of resonance assignments between spectra of different types and different dimensionality, including projected, reduced-dimensionality triple-resonance experiments.

Calibration of the angular dependence of the amide proton-Cα proton coupling constants, 3JHNα, in a globular protein: use of 3JHnα for identification of helical secondary structure

Abstract The vicinal amide proton-C α proton spin-spin coupling constants, 3 J HNα in the globular protein basic pancreatic trypsin inhibitor (BPTI) have been measured using phase-sensitive correlated spectroscopy at high digital resolution. In conjunction with the crystal structure of BPTI, these data were used to calibrate the correlation between 3 J HN α and the dihedral angle φ. The resulting “BPTI curve” is 3 J HN α = 6.4 cos 2 θ − 1.4 cos θ + 1.9 (θ = ¦φ − 60° ¦) . It is further shown that measurement of the spin-spin couplings 3 J HN α presents an independent, reliable method for identification of the location of helical structure in the amino acid sequence of proteins.

Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA

A novel procedure for efficient computation of three-dimensional protein structures from nuclear magnetic resonance (n.m.r.) data in solution is described, which is based on using the program DIANA in combination with the supporting programs CALIBA, HABAS and GLOMSA. The first part of this paper describes the new programs DIANA. CALIBA and GLOMSA. DIANA is a new, fully vectorized implementation of the variable target function algorithm for the computation of protein structures from n.m.r. data. Its main advantages, when compared to previously available programs using the variable target function algorithm, are a significant reduction of the computation time, and a novel treatment of experimental distance constraints involving diastereotopic groups of hydrogen atoms that were not individually assigned. CALIBA converts the measured nuclear Overhauser effects into upper distance limits and thus prepares the input for the previously described program HABAS and for DIANA. GLOMSA is used for obtaining individual assignments for pairs of diastereotopic substituents by comparison of the experimental constraints with preliminary results of the structure calculations. With its general outlay, the presently used combination of the four programs is particularly user-friendly. In the second part of the paper, initial results are presented on the influence of the novel DIANA treatment of diastereotopic protons on the quality of the structures obtained, and a systematic study of the central processing unit times needed for the same protein structure calculation on a range of different, commonly available computers is described.

Polypeptide secondary structure determination by nuclear magnetic resonance observation of short proton-proton distances

The use of proton-proton nuclear Overhauser enhancement (NOE) distance information for identification of polypeptide secondary structures in non-crystalline proteins was investigated by stereochemical studies of standard secondary structures and by statistical analyses of the secondary structures in the crystal conformations of a group of globular proteins. Both regular helix and beta-sheet secondary structures were found to contain a dense network of short 1H-1H distances. The results obtained imply that the combined information on all these distances obtained from visual inspection of the two-dimensional NOE (NOESY) spectra is sufficient for determination of the helical and beta-sheet secondary structures in small globular proteins. Furthermore, cis peptide bonds can be identified from unique, short sequential proton-proton distances. Limitations of this empirical approach are that the exact start or end of a helix may be difficult to define when the adjoining residues form a tight turn, and that unambiguous identification of tight turns can usually be obtained only in the hairpins of antiparallel beta-structures. The short distances between protons in pentapeptide segments of the different secondary structures have been tabulated to provide a generally applicable guide for the analysis of NOESY spectra of proteins.

NMR characterization of the full-length recombinant murine prion protein, mPrP(23–231)

The recombinant murine prion protein, mPrP(23–231), was expressed in E. coli with uniform 15N‐labeling. NMR experiments showed that the previously determined globular three‐dimensional structure of the C‐terminal domain mPrP(121–231) is preserved in the intact protein, and that the N‐terminal polypeptide segment 23–120 is flexibly disordered. This structural information is based on nearly complete sequence‐specific assignments for the backbone amide nitrogens, amide protons and α‐protons of the polypeptide segment of residues 121–231 in mPrP(23–231). Coincidence of corresponding sequential and medium‐range nuclear Overhauser effects (NOE) showed that the helical secondary structures previously identified in mPrP(121–231) are also present in mPrP(23–231), and near‐identity of corresponding amide nitrogen and amide proton chemical shifts indicates that the three‐dimensional fold of mPrP(121–231) is also preserved in the intact protein. The linewidths in heteronuclear 1H–15N correlation spectra and 15N{1H}‐NOEs showed that the well structured residues 126–230 have correlation times of several nanoseconds, as is typical for small globular proteins, whereas correlation times shorter than 1 nanosecond were observed for all residues of mPrP(23–231) outside of this domain.

Prion (PrPSc)-specific epitope defined by a monoclonal antibody

Prions are infectious particles causing transmissible spongiform encephalopathies (TSEs). They consist, at least in part, of an isoform (PrPSc) of the ubiquitous cellular prion protein (PrPC). Conformational differences between PrPCand PrPScare evident from increased β-sheet content and protease resistance in PrPSc(refs 1,2,3). Here we describe a monoclonal antibody, 15B3, that can discriminate between the normal and disease-specific forms of PrP. Such an antibody has been long sought as it should be invaluable for characterizing the infectious particle as well as for diagnosis of TSEs such as bovine spongiform encephalopathy (BSE) or Creutzfeldt–Jakob disease (CJD) in humans. 15B3 specifically precipitates bovine, murine or human PrPSc, but not PrPC, suggesting that it recognizes an epitope common to prions from different species. Using immobilized synthetic peptides, we mapped three polypeptide segments in PrP as the 15B3 epitope. In the NMR structure of recombinant mouse PrP, segments 2 and 3 of the 15B3 epitope are near neighbours in space, and segment 1 is located in a different part of the molecule. We discuss models forthe PrPSc-specific epitope that ensure close spatial proximity of all three 15B3 segments, either by intermolecular contacts in oligomeric forms of the prion protein or by intramolecular rearrangement.