3D structure determination of a protein in living cells using paramagnetic NMR spectroscopy

Abstract

Determining the three-dimensional structure of a protein in living cells remains particularly challenging. We demonstrated that the integration of site-specific tagging proteins and GPS-Rosetta calculations provides a fast and effective way of determining the structures of proteins in living cells, and in principle the interactions and dynamics of protein-ligand complexes.

Improved in-cell structure determination of proteins at near-physiological concentration

“…Recently, structure determinations of GB1 in Xenopus laevis oocyte were reported using exclusively pseudocontact shift and residual dipolar coupling data3738. The protein structure analysis in eukaryotic cells is instructive regarding applications in drug discovery and medical science.…”

A Sterically Overcrowded, Isopropyl‐Substituted, Lanthanide‐Chelating Tag for Protein Pseudocontact Shift NMR Spectroscopy: Synthesis of its Macrocyclic Scaffold and Benchmarking on Ubiquitin S57 C and hCA II S166 C

“…Pseudocontact shifts (PCS) and residual dipolar couplings (RDC) generated by lanthanide‐chelating tags (LCT) yield valuable structural restraints for the analysis of structure, dynamics, and ligand‐binding of proteins in solution . In order to access valuable structural restraints by PCS measurements, stereospecifically methyl‐substituted 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA)‐based chelators present a suitable scaffold .…”

“…Pseudocontact shifts (PCS) and residual dipolar couplings (RDC) generated by lanthanide-chelatingt ags (LCT) yield valuable structural restraints for the analysis of structure, dynamics, and ligand-binding of proteins in solution. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] In order to access valuable structuralr estraintsb yP CS measurements, stereospecifically methyl-substituted1 ,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-based chelators present a suitable scaffold. [9,[19][20][21][22] As investigated by Ranganathane tal., the methyl substituents on the nitrogen-containingm acrocycle in al anthanide complex adopt an equatorial-upperp osition, [23,24] that is, the substituents pointa way from the metal centre in order to accommodate the metal ion in the ligand cavity.I norder to obtain only one signal set in 1 H- 15 NHSQC experiments and to induces trong paramagnetic effects fort he investigation of biomacromolecules in solution, it is mandatory to lock not only the 12-membered ring, [9] but also the pendant arms of the chelator,i no ne conformation.A ss hown by Joss et al,T m-and Dy-DOTA-M8-(4R,4S)-SSPy adopt a L(dddd)c onformation,r esulting in as ingle set of peaks in 1 H- 15 NH SQC spectra.…”

NMR of Macromolecular Assemblies and Machines at 1 GHz and Beyond: New Transformative Opportunities for Molecular Structural Biology

“…The same protocol was subsequently applied to structure determination of GB1 at 250 μM concentration, closer to physiological cellular concentrations (177). Another recent study presented structure determination of GB1 with a paramagnetic tag for PCS structural restraints in eukaryotic cells (178). Enhanced sensitivity from ultrahigh magnetic fields will likely increase the efficiency of in vivo structure determination of low concentration proteins.…”