Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high‐throughput structural studies on proteins (original) (raw)
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Annual Review of Biophysics and Biophysical Chemistry, 1991
Over the past 5 to 10 years, NMR (nuclear magnetic resonance) has developed into a powerful method for determining three-dimensional structures of small proteins of about 100 residues or less. The principal geometric information used in these structure determinations resides in short (< 5 ~) approximate interproton distance restraints derived from the observation of nuclear Overhauser effects (NOE) that are proportional to r-6 (97). The stimulus for this rapid progress has stemmed from one technological and two methodological advances, in particular, the advent of high-field NMR spectrometers (500 600 MHz) and the development a whole array of two-dimensional (2D) NMR experiments and of algorithms for converting NMR-derived interproton distances into cartesian coordinates. The basic strategy for NMR structure determination is relatively straightforward and can be divided into three stages: (a) sequential assignment of backbone and side-chain ~H resonances using experiments that demonstrate through-bond (scalar) and through-space (< 5 ~) nectivities; (b) identification of as many through-space nuclear Overhauser (NOE) connectivities as possible that yield a large set of approximate interproton-distance restraints; and (c) calculation of three-dimensional structures on the basis of these distance restraints, supplemented if possible by some torsion-angle restraints derived from coupling constants. The theoretical basis of two-dimensional NMR has been discussed in detail in a book by Ernst et al (47), and the application of these methods to protein structure determination has been comprehensively reviewed (5, 27, 28, 59, 115, 116). However, several exciting developments from over the past year promise to extend the NMR methodology both with respect to the size of proteins that can be studied and to the accuracy and precision of the structure determinations. These include the extension of 2D NMR into 3D and 4D NMR and the use of systematic database searches to obtain numerous torsion-angle restraints on the basis of the experimental NMR data. In
Journal of Chemical Information and Modeling, 1997
A sequential assignment protocol for proteins was developed using heteronuclear 3D NMR. The protocol consists of an amino acid type recognition algorithm and a primary sequence mapping algorithm. The former measures the similarity between each detected spin pattern and 20 standard amino acid coupling patterns. Both chemical shift and topologically likeness are considered. The mapping algorithm uses the amino acid type information to direct detected polypeptides to proper position onto protein primary sequence. The assignment protocol can be applied to spin systems generated by many different approaches. We designed a few computer programs to derive a protein's backbone and side chain spin systems using heteronuclear 3D NMR. The results was then input to the sequential assignment protocol. All of the algorithms were tested on NMR data of a 90-residue N-domain of chicken skeletal troponin-C.
Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra
Nature communications, 2018
Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6-10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins ...
Journal of Biomolecular NMR, 1998
We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K.(1996) J. Am. Chem. Soc., 118, 8146–8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments ...
Robust structure-based resonance assignment for functional protein studies by NMR
Journal of Biomolecular NMR, 2010
High-throughput functional protein NMR studies, like protein interactions or dynamics, require an automated approach for the assignment of the protein backbone. With the availability of a growing number of protein 3D structures, a new class of automated approaches, called structure-based assignment, has been developed quite recently. Structurebased approaches use primarily NMR input data that are not based on J-coupling and for which connections between residues are not limited by through bonds magnetization transfer efficiency. We present here a robust structure-based assignment approach using mainly H N -H N NOEs networks, as well as 1 H-15 N residual dipolar couplings and chemical shifts. The NOEnet complete search algorithm is robust against assignment errors, even for sparse input data. Instead of a unique and partly erroneous assignment solution, an optimal assignment ensemble with an accuracy equal or near to 100% is given by NOEnet. We show that even low precision assignment ensembles give enough information for functional studies, like modeling of protein-complexes. Finally, the combination of NOEnet with a low number of ambiguous J-coupling sequential connectivities yields a high precision assignment ensemble. NOEnet will be available under: http://www.icsn. cnrs-gif.fr/download/nmr.
Biochemistry, 2001
Sequence specific resonance assignment is the primary requirement for all investigations of proteins by NMR methods. In the present postgenomic era where structural genomics and protein folding have occupied the center stage of NMR research, there is a high demand on the speed of resonance assignment, whereas the presently available methods based either on NOESY or on some triple-resonance experiments are rather slow. They also have limited success with unfolded proteins because of the lack of NOEs, and poor dispersion of amide and carbon chemical shifts. This paper describes an efficient approach to rapid resonance assignment that is suitable for both folded and unfolded proteins, making use of the triple-resonance experiments described recently [HNN and HN(C)N]. It has three underlying principles. First, the experiments exploit the 15 N chemical shift dispersions which are generally very good for both folded and unfolded proteins, along two of the three dimensions; second, they directly display sequential amide and 15 N correlations along the polypeptide chain, and third, the sign patterns of the diagonal and the sequential peaks originating from any residue are dependent on the nature of the adjacent residues, especially the glycines and the prolines. These lead to so-called "triplet fixed points" which serve as starting points and/or check points during the course of sequential walks, and explicit side chains assignment becomes less crucial for unambiguous backbone assignment. These features significantly enhance the speed of data analysis, reduce the amount of experimentation required, and thus result in a substantially faster and unambiguous assignment. Following the amide and 15 N assignments, the other proton and carbon assignments can be obtained in a straightforward manner, from the well-established three-dimensional triple-resonance experiments. We have successfully tested the new approach with different proteins in the molecular mass range of 10-22 kDa, and for illustration, we present here the backbone results on the HIV-1 protease-tethered dimer (molecular mass ∼ 22 kDa), both in the folded and in the unfolded forms, the two ends of the folding funnel. We believe that the new assignment approach will be of great value for both structural genomics and protein folding research by NMR.