Gd(III) Chelates as NMR Probes of Protein–Protein Interactions. Case Study: Rubredoxin and Cytochrome<i>c</i><sub>3</sub>

Almeida, Rui M.; Geraldes, Carlos F. G. C.; Pauleta, Sofia R.; Moura, José J. G.

doi:10.1021/ic200858c

Cited by 16 publications

(12 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Potential binding site of [Ln(L) 3 ] n − ( n =3 for L 1 , L 2 , L 3 , and L 5 ; n =6 for L 4 ) in ubiquitin : Because the paramagnetic relaxation enhancement (PRE) is very sensitive parameter that is used to detect the binding interface of a protein, 6b, c, d residues that experience strong PREs are generally located close to the paramagnetic center. To determine the binding site of the [Ln(L) 3 ] n − complexes, we performed PRE measurements by using [Gd(DPA) 3 ] 3− , [Gd(L 3 ) 3 ] 3− , and [Gd(L 4 ) 3 ] 6− .…”

Section: Resultsmentioning

confidence: 99%

“…[Ln(DPA) 3 ] 3− was first reported to bind specifically to a protein that had a positively charged surface and significant PCS values were observed for a number of proteins 6a. As a consequence, this noncovalent interaction was used to generate PREs 6b–d. Because DPA is symmetrical, the obtained paramagnetic Δ χ tensors are rather similar for a number of paramagnetic lanthanide ions.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Noncovalent Tagging Proteins with Paramagnetic Lanthanide Complexes for Protein Study

Wei

Yang

et al. 2013

Chemistry A European J

View full text Add to dashboard Cite

The site-specific labeling of proteins with paramagnetic lanthanides offers unique opportunities for NMR spectroscopic analysis in structural biology. Herein, we report an interesting way of obtaining paramagnetic structural restraints by employing noncovalent interaction between a lanthanide metal complex, [Ln(L)3](n-) (L=derivative of dipicolinic acid, DPA), and a protein. These complexes formed by lanthanides and DPA derivatives, which have different substitution patterns on the DPA derivatives, produce diverse thermodynamic and paramagnetic properties when interacting with proteins. The binding affinity of [Ln(L)3](n-) with proteins, as well as the determined paramagnetic tensor, are tunable by changing the substituents on the ligands. These noncovalent interactions between [Ln(L)3](n-) and proteins offer great opportunities in the tagging of proteins with paramagnetic lanthanides. We expect that this method will be useful for obtaining multiple angles and distance restraints of proteins in structural biology.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Noncovalent Tagging Proteins with Paramagnetic Lanthanide Complexes for Protein Study

Wei

Yang

et al. 2013

Chemistry A European J

View full text Add to dashboard Cite

show abstract

“…Lanthanide incorporation is a promising strategy to expand the utility of fluorescence and NMR in examining large biopolymers, offering the ability to evaluate protein dynamics [5], protein conformational changes [24, 44], and interactions of larger proteins than have previously been studied [7, 22]. However, the requirement for site-specific incorporation, coupled with the need for a lanthanide chelator, have limited its use.…”

Section: Discussionmentioning

confidence: 99%

“…However, the LBT’s rather large size, which may interfere with proper folding and/or function of the target protein, has prompted efforts to pursue small molecule lanthanide chelators [20–23]. Small molecule lanthanide chelators such as ethylenediaminetetraacetic acid (EDTA) and its derivatives 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and diethylenetriaminepentaacetic acid (DTPA) exhibit attomolar affinity for lanthanide metals and have been used for decades for their superior luminescence intensities [11, 24], and more recently, for NMR experiments [22].…”

Section: Introductionmentioning

confidence: 99%

Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

et al. 2019

View full text Add to dashboard Cite

The lanthanides (Ln 3+ ), or rare earth elements, have proven to be useful tools for biomolecular NMR, X-ray crystallographic, and fluorescence analyses due to their unique 4 f orbitals. However, their utility in biological applications has been limited because site-specific incorporation of a chelating element is required to ensure efficient binding of the free Ln 3+ ion. Additionally, current Ln 3+ chelator syntheses complicate efforts to directly incorporate Ln 3+ chelators into proteins as the multi-step processes and a reliance on organic solvents promote protein denaturation and aggregation which are generally incompatible with direct incorporation into the protein of interest. To overcome these limitations, herein we describe a two-step aqueous synthesis of a small molecule lanthanide chelating agent amenable to site-specific incorporation into a protein using copper-free click chemistry with unnatural amino acids. The bioconjugate combines a diethylenetriaminepentaacetic acid (DTPA) chelating moiety with a clickable dibenzylcyclooctyne-amine (DBCO-amine) to facilitate the reaction with an azide containing unnatural amino acid. Incorporating the DBCO-amine avoids the use of the cytotoxic Cu 2+ ion as a catalyst. The clickable lanthanide chelator (CLC) reagent reacted readily with p -azidophenylalanine (paF) without the need of a copper catalyst, thereby demonstrating proof-of-concept. Implementation of the orthogonal click chemistry reaction has the added advantage that the chelator can be used directly in a protein labeling reaction, without the need of extensive purification. Given the inherent advantages of Cu 2+ -free click chemistry, aqueous synthesis, and facile labeling, we believe that the CLC will find abundant use in both structural and biophysical studies of proteins and their complexes.

show abstract

“…sPRE experiments based on the interactions of the two probes with the surface of the proteins allowed to gain information about charged patches on the protein surface and to characterize the binding site in the protein complex. 14 As each of the paramagnetic effects has its peculiarities, it is emphasized that combining restraints based on PCSs, RDCs and PREs is a safeguard against biased results. For example, structure calculations based only on PREs may be prone to bias toward minor states, because the PREs are so sensitive to those.…”

Section: The New Toolboxmentioning

confidence: 99%

Protein–Protein Interactions

Ubbink

Savino

2018

Paramagnetism in Experimental Biomolecular NMR

View full text Add to dashboard Cite

Paramagnetic NMR methods are excellently suited for the study of protein-protein complexes in solution. Intermolecular pseudocontact shifts (PCSs), residual dipolar couplings (RDCs) and paramagnetic relaxations enhancements (PRE) can be used, ideally in combination, for docking proteins and determining their orientation in the complex. PCSs can be used for breaking the structure symmetry in dimer complexes. PCSs also can be applied to detect structural differences in proteins and protein complexes in solution in comparison to crystal structures. RDCs are sensitive to the degree of alignment of both partners in a protein complex and are thus very useful to detect dynamics within complexes. PREs can detect states in which nuclei approach a paramagnetic centre closely, even if it exists only for a small fraction of the time. Thus, PREs are used to detect minor states and characterize ensembles.PRE studies have been the foundation for characterizing encounter states and the process of protein complex formation. In weak complexes, such as found in electron transfer chains, proteins can be in an encounter state for a large fraction of the complex lifetime.Paramagnetic NMR tools thus have found many applications for studying protein complexes, and more may be on the horizon.

show abstract

Gd(III) Chelates as NMR Probes of Protein–Protein Interactions. Case Study: Rubredoxin and Cytochromec₃

Cited by 16 publications

References 55 publications

Noncovalent Tagging Proteins with Paramagnetic Lanthanide Complexes for Protein Study

Noncovalent Tagging Proteins with Paramagnetic Lanthanide Complexes for Protein Study

Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

Protein–Protein Interactions

Contact Info

Product

Resources

About

Gd(III) Chelates as NMR Probes of Protein–Protein Interactions. Case Study: Rubredoxin and Cytochromec3

Cited by 16 publications

References 55 publications

Noncovalent Tagging Proteins with Paramagnetic Lanthanide Complexes for Protein Study

Noncovalent Tagging Proteins with Paramagnetic Lanthanide Complexes for Protein Study

Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

Protein–Protein Interactions

Contact Info

Product

Resources

About

Gd(III) Chelates as NMR Probes of Protein–Protein Interactions. Case Study: Rubredoxin and Cytochromec₃