Structure determination of uniformly 13C, 15N labeled protein using qualitative distance restraints from MAS solid-state 13C-NMR observed paramagnetic relaxation enhancement

Abstract

Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the Xray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library. KeywordsSolid-state NMR Magic-angle spinning Protein structure Paramagnetic relaxation enhancement CS-Rosetta Electronic supplementary materialThe online version of this article

High Accuracy Protein Structures from Minimal Sparse Paramagnetic Solid‐State NMR Restraints

“…Even when using the complete set of PREs,the convergence properties of the Xplor-NIH based approach were relatively poor.Combining our semi-quantitative MELD approach with the same dataset results in amuch tighter structural ensemble ( Figure 2, "Full"), and ar epresentative model that is 0.9 from native with excellent sidechain packing ( Figure 3). Following aclosely related experimental approach, Tamaki et al [14] used aC S-Rosetta [34] based protocol to fold GB1 to within 1.5 of the native structure using three paramagnetic mutants.The use of PRE data from single Mn 2+ -EDTAa nalogs resulted in structural models with RMSDs in the 4.5-8 regime. Following aclosely related experimental approach, Tamaki et al [14] used aC S-Rosetta [34] based protocol to fold GB1 to within 1.5 of the native structure using three paramagnetic mutants.The use of PRE data from single Mn 2+ -EDTAa nalogs resulted in structural models with RMSDs in the 4.5-8 regime.…”

“…[11,14] Collectively,these studies highlight the considerable promise of such approaches.A tt he same time,t hey underscore the urgent need for more effective approaches toward generating accurate structural models based on paramagnetic restraints. [11,14] Collectively,these studies highlight the considerable promise of such approaches.A tt he same time,t hey underscore the urgent need for more effective approaches toward generating accurate structural models based on paramagnetic restraints.…”

Festkörper‐NMR‐ und EPR‐Spektroskopie an Mn2+‐substituierten ATP‐angetriebenen Proteinmaschinen

“…Die Resonanzzuordnungen wurden aus Lit. [21] PRE-Effekte skalieren mit r À6 ,wobei r den Abstand zwischen dem Metallzentrum und dem Kern im Protein repräsentiert. Drei Ty pen von Resonanzen kçnnen auf Basis der 2D-Spektren unterschieden werden: Reste,d ie nicht durch Mn 2+ beeinflusst werden (z.…”

“…Jede dieser Domänen enthält Walker-A-Walker-B-Motive,w obei ein Aspartat das Mg 2+ koordiniert. [17][18][19][20][21] Dies ermçglicht eine Unterscheidung zwischen der Bindungsstelle und von dieser entfernten Te ilen des Systems.A ufgrund der vergleichbaren Koordinationseigenschaften, Ladungen und Radien von Mg 2+ -und Mn 2+ -Ionen wird die Funktion des Proteins in den meisten Fällen beibehalten (siehe Lit. Um die Nukleotid-gebundenen Zustände zu imitieren, werden schlecht hydrolysierbare ATP-Analoga wie Adenylylimidodiphosphat (AMP-PNP) verwendet, die mit einer hohen AffinitätanHpDnaB binden.…”

Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy

“…The precision of these PRE-based distance measurements, as well as their utility for protein structure determination is expected to further improve by using Cu(II)-binding tags that are more compact and rigid relative to the EDTA-type tag employed in the present study, 15,22 as well as multiple protein samples containing the paramagnetic tags in several different locations. 13,16 …”