Solution Structure of the Paramagnetic Complex of the N-Terminal Domain of Calmodulin with Two Ce3+ Ions by 1H NMR,

Bentrop, Detlef; Bertini, Ivano; Cremonini, Mauro Andrea; Forsén, Sture; Luchinat, Claudio; Malmendal, Anders

doi:10.1021/bi971022+

Cited by 101 publications

(79 citation statements)

References 45 publications

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“…3). We then calculated the PCSs for IL1b based on the hypothesis that Dv-tensors of IL1b-R2 and -S2 are additive (Bentrop et al 1997) in IL1b-S2R2. The correlation of the calculated PCSs to the experimental PCSs obtained for IL1b-S2R2 is remarkably good with an R 2 of 0.959 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

et al. 2015

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We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1b (IL1b) and measured 1 H, 15 Npseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Dv-tensors associated with each Tm 3? -loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein-protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1b-S2R2 and the respective single-loop-LBT constructs IL1b-S2, IL1b-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd 3? as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron-Electron Dipolar Resonance.

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Section: Resultsmentioning

confidence: 99%

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

et al. 2015

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“…60 The N60D mutation and the lanthanide substitution do not lead to any significant structural changes in the region flanking the metal coordination site. 52,60,78 PCSs of H N , N nuclei, and RDCs of the H N −N pairs for six paramagnetic lanthanide (CaLn) N (Ca 2 ) C CaMN60D forms in complex with IQ peptide (Ln = Tb, Dy, Ho, Er, Tm, Yb) were measured using 1 H, 15 N HSQC and IPAP spectra, respectively (Figure 1).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Interdomain Dynamics Explored by Paramagnetic NMR

et al. 2013

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An ensemble-based approach is presented to explore the conformational space sampled by a multidomain protein showing moderate interdomain dynamics in terms of translational and rotational motions. The strategy was applied on a complex of calmodulin (CaM) with the IQ-recognition motif from the voltage-gated calcium channel Ca v 1.2 (IQ), which adopts three different interdomain orientations in the crystal. The N60D mutant of calmodulin was used to collect pseudocontact shifts and paramagnetically induced residual dipolar couplings for six different lanthanide ions. Then, starting from the crystal structure, pools of conformations were generated by free MD. We found the three crystal conformations in solution, but four additional MD-derived conformations had to be included into the ensemble to fulfill all the paramagnetic data and cross-validate optimally against unused paramagnetic data. Alternative approaches led to similar ensembles. Our "ensemble" approach is a simple and efficient tool to probe and describe the interdomain dynamics and represents a general method that can be used to provide a proper ensemble description of multidomain proteins.

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“…Ligand donor atoms Asp-9 O ␦1 , Asp-11 O ␦1 , Asp-13 O ␦1 , Thr-15 O, and both Glu-20 O were constrained during the annealing process (24) to distances from the metal representing consensus lanthanide(III)-EF-hand ligation (19,25). However, the 3D coordination geometry (O-Ln-O angles) around the metal were not defined in any way.…”

Section: Experimental Methodsmentioning

confidence: 99%

Lanthanide-binding helix-turn-helix peptides: Solution structure of a designed metallonuclease

Welch

Kearney

Franklin

2003

Proc. Natl. Acad. Sci. U.S.A.

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A designed lanthanide-binding chimeric peptide based on the strikingly similar geometries of the EF-hand and helix-turn-helix (HTH) motifs was investigated by NMR and CD spectroscopy and found to retain the same overall solution structure of the parental motifs. CD spectroscopy showed that the 33-mer peptide P3W folds on binding lanthanides, with an increase in ␣-helicity from 20% in the absence of metal to 38% and 35% in the presence of excess Eu(III) and La(III) ions, respectively. The conditional binding affinities of P3W for La(III) (5.9 ؎ 0.3 M) and for Eu(III) (6.2 ؎ 0.3 M) (pH 7.8, 5 mM Tris) were determined by tryptophan fluorescence titration. The La(III) complex of peptide P3, which differs from P3W by only one Trp-to-His substitution, has much less signal dispersion in the proton NMR spectra than LaP3W, indicating that the Trp residue is a critical hydrophobic anchor for maintaining a well-folded helixturn-helix structure. A chemical-shift index analysis indicates the metallopeptide has a helix-loop-helix secondary structure. A structure calculated by using nuclear Overhauser effect and other NMR constraints reveals that P3W not only has a tightly folded metalbinding loop but also retains the ␣؊␣ corner supersecondary structure of the parental motifs. Although the solution structure is undefined at both the N and C termini, the NMR structure confirms the successful incorporation of a metal-binding loop into a HTH sequence. One of the great promises of de novo protein design is the possibility of incorporating new reactivity or selectivity into a protein matrix for specific chemical targets or to advance biological understanding. The redesign of a known protein fold is one powerful approach to achieving new function, and the inclusion of metal sites that could confer a range of enzymatic activities is particularly intriguing. Designed metalloproteins have the potential to promote natural enzymatic reactions on unnatural substrates or even to achieve reactivity through mechanisms completely unprecedented in biology. However, to interpret and evaluate their activity and to improve subsequent designs, the resultant constructs must retain a predictable well-folded structure. A structurally well characterized robust scaffold is therefore fundamental to the design of minimalist enzymes.In recent years, several approaches have been taken to engineer novel metalloproteins, including building a metal site and scaffold completely from first principles (1-5), grafting ligands onto known protein scaffolds by point mutations that were identified through modeling or a vector-based algorithm such as DENZYMER (5-9), or redesigning natural metalloproteins for potential new reactivity (10, 11). Alternately, native metal-binding sequences may be used in new contexts (12-14), and we have recently reported the de novo design (or more correctly, redesign) of metallopeptide nucleases based on this premise (12, 15).The structural similarity in their natural protein contexts of the helix-turn-helix (HTH), a DNA-binding motif c...

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Solution Structure of the Paramagnetic Complex of the N-Terminal Domain of Calmodulin with Two Ce³⁺ Ions by ¹H NMR^,

Cited by 101 publications

References 45 publications

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

Interdomain Dynamics Explored by Paramagnetic NMR

Lanthanide-binding helix-turn-helix peptides: Solution structure of a designed metallonuclease

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