The use of a ditopic Gd(III) paramagnetic probe for investigating α-bungarotoxin surface accessibility (2023)

Table of Contents
Journal of Inorganic Biochemistry Abstract Graphical abstract Highlights Introduction Section snippets Sample preparation and NMR measurements Gd2L7 structural characterization Discussion Abbreviations Acknowledgments References (37) Biophys. J. J. Magn. Reson. Adv. Inorg. Chem. Comp. Physiol. Commun. Biophys. J. Biochem. Biophys. Res. Commun. J. Mol. Biol. Am. J. Med. Sci. Prog. Nucl. Magn. Reson. Spectrosc. Biochim. Biophys. Acta ChemBioChem Nucleic Acids Res. J. Am. Chem. Soc. Chemistry J. Phys. Chem. A J. Am. Chem. Soc. Investig. Radiol. Cited by (11) Solvent paramagnetic relaxation enhancement as a versatile method for studying structure and dynamics of biomolecular systems Probing Surfaces in Dynamic Protein Interactions Investigations into the killing activity of an antimicrobial peptide active against extensively antibiotic-resistant K. pneumon iae and P. aeruginosa Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies Paramagnetic Chemical Probes for Studying Biological Macromolecules NMR techniques in studying water in biotechnological systems Recommended articles (6) First in vivo MRI study on theranostic dendrimersomes The amyloid precursor protein (APP) intracellular domain regulates translation of p44, a short isoform of p53, through an IRES-dependent mechanism Efficient digestion of chitosan using chitosanase immobilized on silica-gel for the production of multisize chitooligosaccharides Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1) Non-Heme Iron-Dependent Enzymes That Cleave Carbon-Carbon Bonds During Phosphonate Biosynthesis Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides

Journal of Inorganic Biochemistry

Volume 112,

July 2012

, Pages 25-31

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Protein surface accessibility is a critical parameter which drives all intermolecular interaction processes. In this respect a big deal of information has been derived by analyzing paramagnetic perturbation profiles obtained from NMR protein spectra, particularly in the case that the effects due to different soluble paramagnets can be compared. Here Gd2L7, a neutral ditopic paramagnetic NMR probe, has been characterized in terms of structure and relaxivity and its paramagnetic perturbations on α-bungarotoxin CαH signals in 1H–13C HSQC (heteronuclear single quantum coherence) spectra have been analyzed. Then, these signal attenuations have been compared with the ones previously obtained in the presence of GdDTPA-BMA (gadolinium(III) diethylenetriamine-N,N,N′,N'″,N″-pentaacetate-bis(methylamide)). In spite of the different molecular size and shape, for the two probes a common pathway of approach to the α-bungarotoxin surface can be observed with an equally enhanced access of both GdDTPA-BMA and Gd2L7 toward the protein surface side where residues involved in the receptor binding are located. The different residence times of the water molecule directly coordinated by the Gd(III) ion measured for the two paramagnets account for the reduced broadening of water signal in the presence of the ditopic probe at equivalent gadolinium concentration. These features make Gd2L7 a very suitable probe for investigating protein surface accessibility of complex protein systems.

Graphical abstract

Gd2L7, a ditopic gadolinium paramagnet, was tested for α-bungarotoxin hot spots mapping. Gd2L7 paramagnetic relaxation features make it more suitable than GdDTPA-BMA for NMR studies on protein surface accessibility. Long residence time of Gd2L7 coordinated water molecule yields good water signal suppression, but low efficiency as MRI contrast agent.

The use of a ditopic Gd(III) paramagnetic probe for investigating α-bungarotoxin surface accessibility (3)
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► Gd2L7 is a ditopic paramagnet suitable for hot spots mapping from NMR studies. ► Long residence time of Gd-coordinated water limits broadening of water NMR signal. ► Efficient MRI contrast agents hardly are good probes for protein hot spots mapping. ► α-Bungarotoxin binding site is similarly approached by Gd2L7 and GdDTPA-BMA.


The use of proteins as molecular devices in bionanotechnology requires a detailed knowledge of the mechanisms of their interactions with the surrounding molecular environment. It is apparent, indeed, that protein surface accessibility controls all the biological processes determined by protein–protein, protein–ligand and protein–nucleic acid interactions. Thus, mapping protein surface accessibility can provide a solid experimental basis to predict surface hot spots and, hence, to design bionanodevices with suitable mutants.

Nuclear magnetic resonance (NMR) studies on the effects induced by soluble paramagnetic probes, such as aminoxyl spin-labels [1], [2], [3], gadolinium complexes [4], [5] and molecular oxygen [6], on protein and RNA NMR signals have provided a wealth of information about the complex dynamics contributing to surface accessibility. The fact that paramagnetic probes might be involved in biased approaches toward specific amino acid side chains or structural determinants has been considered and, consequently, the use of more than one probe has been suggested to enhance the resolution of this kind of studies [7], [8].

Some interference to a purely random approach of TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) to the protein surface, due to some hydrogen bonding between the N-oxyl moiety of the probe and protein backbone amide groups, has been suggested by comparing the paramagnetic profiles induced by different probes [7]. Relaxometric techniques have yielded no evidence of strong interactions of GdDTPA-BMA (gadolinium(III) diethylenetriamine-N,N,N′,N″,N″-pentaacetate-bis(methylamide)) with specific molecular sites, even in the crowded molecular environments typical of biological fluids [9], suggesting that the latter paramagnet is well suited for accurate mapping of surface accessibility [4].

To overcome risks from toxicity [10], [11] and reduced MRI (magnetic resonance imaging) contrasting activity at high magnetic fields [12] of conventional paramagnetic probes, engineering of a large variety of new paramagnets is required. NMR investigations on protein surface accessibility can take advantage of the latter MRI needs, broadening the repertoire of neutral soluble paramagnets to study the interaction between proteins and small molecules.

In the present study a ditopic neutral complex of Gd(III) with L7 macrocycle (see Fig.1 and caption for structure and full name), henceforth called Gd2L7, was used for investigating the role of size and hydrophobicity of paramagnetic probes in defining protein surface accessibility. Paramagnetic attenuations induced by Gd2L7 on well resolved CαH signals of 1H–13C heteronuclear single quantum coherence (HSQC) spectra of α-bungarotoxin, α-BTX, have been analyzed. This small neurotoxin, having well characterized structural and dynamic features [13], [14], represents a suitable model system to investigate on specific pathways of approach of a paramagnetic probe to the protein surface. Thus, by using GdDTPA-BMA, a remarkable complementarity between MD (molecular dynamics) predicted low water density and high paramagnetic attenuations has been found [15]. The surface dynamics picture inferred by the latter NMR study results in very good agreement with the recent crystallographic structure of α-BTX bound with its receptor [16]. Indeed, the protein moiety most accessible to GdDTPA-BMA results also to be the binding site with the α1 subunit of the mouse nicotinic acetylcholine receptor. Moreover, in the protein–receptor interface no water molecules are present, consistent with the lack of high water density sites in the α-BTX binding region predicted by MD simulations [15].

In the case that Gd2L7 would confirm the accessibility pattern toward the α-BTX surface which has been already derived from GdDTPA-BMA paramagnetic perturbations, the ditopic probe should be included in the list of molecular tools for understanding the dynamics of protein surface accessibility and, hence, for accurate mapping of protein surface hot spots.

Section snippets

Sample preparation and NMR measurements

α-BTX was obtained from Sigma and used without any further manipulation. Gd2L7 was prepared as previously described [1]. NMR measurements, run at 303K and pH 6.0 to reproduce the experimental conditions of the original structural study of α-BTX, were obtained with a Bruker Avance DRX 600 spectrometer.

The 1H–13C HSQC diamagnetic and paramagnetic spectra were obtained with 160 increments and 192 scans over 2048 data points, with an inter-scan delay of 5.0s and an INEPT (insensitive nuclei

Gd2L7 structural characterization

The X-ray structure of gadolinium complex was solved by single crystal X-ray diffraction analysis. Crystallographic data and details of data collection and refinement are given in Table1. The asymmetric unit contains two crystallographically independent Gd(III) ions linked together through two phenyl groups to construct a centrosymmetric dinuclear secondary building unit of [Gd2C44H60N10O18·18.5(H2O)]. Each gadolinium ion is nine-coordinated and its coordination cage is constituted by three


It has been already shown that the structural interpretation of perturbations induced by soluble and neutral paramagnetic probes is not straightforward [15]. The use of atom depths, rather than accessible surface areas, ASA, has been proposed as a step forward to discuss the extent of paramagnetic relaxation effects, PRE, in terms of protein structure[15]. Atom depth, indeed, reflects more properly than ASA the through-space character of those dipolar interactions between electronic and



Thanks are due to Caterina Bernini for technical assistance. Thanks are also due to the Istituto Toscano Tumori, University of Siena and University of Eastern Piedmont for financial support. W.-T.W. thanks the Hong Kong Research Grants Council (HKU7116/02P) and the University of Hong Kong for financial support. A.P.-L.T. thanks The University of Hong Kong for financial support.

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