Three-Dimensional Protein Structure Determination Using Pseudocontact Shifts of Backbone Amide Protons Generated by Double-Histidine Co²⁺-Binding Motifs at Multiple Sites

Abstract

Pseudocontact shifts (PCS) generated by paramagnetic metal ions contribute highly informative long-range structure restraints that can be measured in solution and are ideally suited to guide structure prediction algorithms in determining global protein folds. We recently demonstrated that PCSs, that are relatively small but of high quality, can be generated by a double histidine (dHis) motif in an -helix, which provides a well-defined binding site for a single Co 2+ ion. Here we show that PCSs of backbone amide protons generated by dHis-Co 2+ motifs positioned in four different -helices of a protein deliver excellent restraints to determine the three-dimensional (3D) structure of a protein in a way akin to the global positioning system (GPS). We demonstrate the approach with GPS-Rosetta calculations of the 3D structure of the C-terminal domain of the chaperone ERp29 (ERp29-C). Despite the relatively small size of the PCSs generated by the dHis-Co 2+ motifs, the structure calculations converged readily. Generating PCSs by the dHis-Co 2+ motif thus presents an excellent alternative to the use of lanthanide tags.

Paramagnetic Solid‐State NMR to Localize the Metal‐Ion Cofactor in an Oligomeric DnaB Helicase

“…Since ATP‐binding is accompanied by the binding of Mg 2+ , [6] substitution of Mg 2+ by paramagnetic ions, such as Mn 2+ or Co 2+ , allows us to introduce paramagnetic probes into the protein assembly. This approach is commonly applied in NMR as well as EPR and paramagnetic effects are often used in structure calculation protocols [7–16] . The oligomeric character of DnaB and thus its inherent size, limit however the use of solution‐state NMR due to the broadening of the lines induced by fast T 2 relaxation for slowly tumbling large proteins (life‐time broadening effects) [17,18] .…”

“…This approach is commonly applied in NMR as well as EPR and paramagnetic effects are often used in structure calculation protocols. [7][8][9][10][11][12][13][14][15][16] The oligomeric character of DnaB and thus its inherent size, limit however the use of solution-state NMR due to the broadening of the lines induced by fast T 2 relaxation for slowly tumbling large proteins (life-time broadening effects). [17,18] Solid-state NMR is not affected by this size limitation and offers also significant advantages compared to X-ray crystallography in that it is wellsuited for the investigation of difficult-(or even impossible)-tocrystallize biomolecular assemblies.…”

The Photocatalyzed Thiol‐ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins

“…Structure calculation by NMR mainly relies on the collection of short‐range distance restraints (up to ∼5–6 Å) provided by the time‐consuming and troublesome analysis of NOESY spectra. The use of long‐range paramagnetic distance restraints (up to ∼40 Å), such as pseudo‐contact shifts (PCS), induced by a paramagnetic ion, has been widely proposed to help in de novo structure determination by NMR or in the refinement of pre‐existing X‐ray structures . In this way, a more reliable model describing the protein in solution can be obtained from the crystal structure …”

A Paramagnetic NMR Spectroscopy Toolbox for the Characterisation of Paramagnetic/Spin-Crossover Coordination Complexes and Metal-Organic Cages

“…Paramagnetic NMR spectroscopy [21][22][23][24][25] is central to many fields from chemical and structural biology for studying the structure, dynamics and interactions of proteins 2,3,26,27 to probing spin-state populations in spincrossover compounds [28][29][30][31][32][33][34] and the structural characterisation of paramagnetic complexes 35,36 and supramolecular architectures. 14,[37][38][39][40][41] However, NMR data acquisition and interpretation in the presence of a paramagnetic centre presents a number of challenges due to the large paramagnetic shifts, short relaxation times and broad linewidths: pulse programs with long or multiple pulses are not suitable since relaxation can occur before data acquisition takes place; 22 uniform excitation is more difficult over the larger spectral range; some signals may be lost completely in the case of very short relaxation times and broad linewidths; 25 structural information usually extracted from the chemical shift and J-coupling is lost.…”