Backbone assignment of proteins with known structure using residual dipolar couplings

Abstract

A prerequisite for NMR studies of protein-ligand interactions or protein dynamics is the assignment of backbone resonances. Here we demonstrate that protein assignment can significantly be enhanced when experimental dipolar couplings (RDCs) are matched to values back-calculated from a known three-dimensional structure. In case of small proteins, the program MARS allows assignment of more than 90% of backbone resonances without the need for sequential connectivity information. For bigger proteins, we show that the combination of sequential connectivity information with RDC-matching enables more residues to be assigned reliably and backbone assignment to be more robust against missing data. Structural or dynamic deviations from the employed 3D coordinates do not lead to an increased error rate in RDC-supported assignment. RDC-enhanced assignment is particularly useful when chemical shifts and sequential connectivity only provide a few reliable assignments.

Regulation of the Plasmodium Motor Complex

“…The chemical shifts of 1 H N , 15 N, 13 C ␣ , 13 C ␤ , and 13 CЈ were obtained from the HNCACB/CBCA(CO)NH and HNCO/ HN(CA) CO experiments; the spectra were analyzed in NMRView, and assignments were made using a combination of the MARS automated assignment program (34) and an in-house assignment module (35 30.66 and 168.61 ms), and the NOE spectrum used a 1 H presaturation period of 5 s. T 1 and T 2 relaxation curves were fit to simple exponential functions using the rate analysis module in NMRView (33), and NOEs are reported as the ratio of intensities of the backbone amide peaks with and without 1 H presaturation. CPMG relaxation dispersion NMR experiments were performed at 14.1 and 18.8 T using the pulse sequences reported by Kay and co-workers (36).…”

Residual Dipolar Couplings: Measurements and Applications to Biomolecular Studies

“…Recently, several algorithms have been developed to assign the resonances of backbone nuclei using either RDCs alone or RDCs in combination with the NMR data used for the traditional approaches. These RDC-based automated assignment algorithms all require that either there exists an a priori structure [144,145,146] or intermediate structures can be determined on-the-fly [147,148,149,150].…”

“…NVR achieved an average assignment accuracy of over 99 % when the chemical shift errors for amide proton 1 H N and amide nitrogen 15 N were set to be, respectively, ±0.01ppm and ±0.1ppm. [154,146], for sequential assignment of backbone resonances based on (a) the chemical shift predication obtained via a secondary structure prediction method, (b) the identification of amino acid types from the chemical shifts for C α , C β and C . MARS requires an 15 N and 13 C doubly-labeled sample and triple-resonance experiments.…”

“…The original MARS algorithm [154] was extended to incorporate RDCs where RDCs are used essentially for filtering out the false positives in connectivity obtained from the original MARS using chemical shift information alone. In case of small proteins and with three RDCs per residue, MARS [146] can assign more than 90% of backbone resonances with an assignment reliability of 56% without the need for sequential connectivity information. For bigger proteins, the combination of sequential connectivity information with RDC-matching enables more residues to be assigned.…”

Quantitative Analysis of Biomolecular NMR Spectra: A Prerequisite for the Determination of the Structure and Dynamics of Biomolecules

“…The use of dipolar couplings to recognize protein structural motifs was proposed [94] in order to be included in annotation in the frame of proteomics projects. The RDCs and the chemical shifts are sufficient [119,120] for the spectral assignment of a protein of known structure, without using any sequential NMR connectivity information. An algorithm called Nuclear Vector Replacement (NVR) [121,122] was introduced to perform the assignment of a protein of known structure, based on RDCs and NOEs.…”