No abstract

No abstract

No abstract

NMR spectroscopy with proteins based on observation of a small number of spins with outstanding spectral properties, which either may be present naturally or introduced by techniques such as site-specific isotope labeling, yielded biologically relevant information on human hemoglobin (M ϭ 65,000) as early as 1969 (1), and subsequently also for significantly larger systems such as Igs (2). In contrast, the use of NMR for de novo structure determination (3, 4) so far has been limited to relatively small molecular sizes, with the largest NMR structure below molecular weight 30,000. Although NMR in structural biology may, for practical reasons of coordinated use with x-ray crystallography (5), focus on smaller molecular sizes also in the future, considerable effort goes into attempts to extend the size limit to bigger molecules (for example, see refs. 6-8). Here we introduce transverse relaxation-optimized spectroscopy (TROSY) and present experimental data and theoretical considerations showing that this approach is capable of significantly reducing transverse relaxation rates and thus overcomes a key obstacle opposing solution NMR of larger molecules (7).At the high magnetic fields typically used for studies of proteins and nucleic acids, chemical shift anisotropy interaction (CSA) of 1 H, 15 N, and 13 C nuclei forms a significant source of relaxation in proteins and nucleic acids, in addition to dipole-dipole (DD) relaxation. This leads to increase of the overall transverse relaxation rates with increasing polarizing magnetic field, B 0 . Nonetheless, transverse relaxation of amide protons in larger proteins at high fields has been reduced successfully by complete or partial replacement of the nonlabile hydrogen atoms with deuterons and, for example, more than 90% of the 15 N, 13 C ␣ , and 1 H N chemical shifts thus were assigned in the polypeptide chains of a protein-DNA complex of size 64,000 (6). TROSY uses spectroscopic means to further reduce T 2 relaxation based on the fact that cross-correlated relaxation caused by DD and CSA interference gives rise to different relaxation rates of the individual multiplet components in a system of two coupled spins 1 ⁄2, I and S, such as the 15 N-1 H fragment of a peptide bond (9, 10). Theory shows that at 1 H frequencies near 1 GHz nearly complete cancellation of all transverse relaxation effects within a 15 N-1 H moiety can be achieved for one of the four multiplet components. TROSY observes exclusively this narrow component, for which the residual linewidth is then almost entirely because of DD interactions with remote hydrogen atoms in the protein. These can be efficiently suppressed by 2 H-labeling, so that in TROSY-type experiments the accessible molecular size for solution NMR studies no longer is primarily limited by T 2 relaxation. TheoryWe consider a system of two scalar coupled spins 1 ⁄2, I and S, with a scalar coupling constant J IS , which is located in a protein molecule. T 2 relaxation of this spin system is dominated by the DD coupling of I and S a...

No abstract

No abstract

A 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.

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 crosspeak 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 crosspeak 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 0022-2836/02/$ -see front matter q 2002 Elsevier Science Ltd. All rights reserved Present address: P. Gü ntert, RIKEN Genomic Sciences Center, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan.E-mail address of the corresponding author: guentert@gsc.r...