Journal of Inorganic Biochemistry
, Pages 25-31
Author links open overlay panel, , , , , , , ,
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.
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.
► 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 , , , gadolinium complexes ,  and molecular oxygen , 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 , .
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 . 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 , suggesting that the latter paramagnet is well suited for accurate mapping of surface accessibility .
To overcome risks from toxicity ,  and reduced MRI (magnetic resonance imaging) contrasting activity at high magnetic fields  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 , , 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 . 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 . 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 .
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.
Sample preparation and NMR measurements
α-BTX was obtained from Sigma and used without any further manipulation. Gd2L7 was prepared as previously described . 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 . 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. Atom depth, indeed, reflects more properly than ASA the through-space character of those dipolar interactions between electronic and
heteronuclear single quantum coherence
insensitive nuclei enhanced by polarization transfer
magnetic resonance imaging
nuclear magnetic resonanceSee AlsoProtocol: Staining Cells with Hoechst or DAPI Nuclear Stains - BiotiumEffects of pharmacological agents on the hypothalamus of Rana pipiens in relation to the control of skin melanophoresα7 nAChRs expressed on antigen presenting cells are insensitive to the conventional antagonists α-bungarotoxin and methyllycaconitineStudies on the hippocampal formation: From basic development to clinical applications: Studies on schizophrenia
nuclear magnetic resonance dispersion
root mean square fluctuation
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.
- A. De Simone et al.
- C.-L. Teng et al.
J. Magn. Reson.
- S. Aime et al.
Adv. Inorg. Chem.
- H.J.C. Berendsen et al.
Comp. Physiol. Commun.
- H. Molinari et al.
- V. Venditti et al.
Biochem. Biophys. Res. Commun.
- A. Bernini et al.
J. Mol. Biol.
- M.A. Perazella et al.
Am. J. Med. Sci.
- A. Bernini et al.
Prog. Nucl. Magn. Reson. Spectrosc.
- A. Bernini et al.
Biochim. Biophys. Acta
Nucleic Acids Res.
J. Am. Chem. Soc.
J. Phys. Chem. A
J. Am. Chem. Soc.
Solvent paramagnetic relaxation enhancement as a versatile method for studying structure and dynamics of biomolecular systems
2022, Progress in Nuclear Magnetic Resonance Spectroscopy
Citation Excerpt :
Gu et al. have developed a spherical and regular agent, TTHA-TMA (triethylenetetraamine hexaacetate trimethylamide), containing 10 coordination sites to the gadolinium ion, thus removing a free coordination site for a water molecule and avoiding enhancing the relaxation of protein backbone resonances through contributions from water-exchangeable protons [60,164]. Furthermore, aiming at the investigation of macromolecular complexes, larger probes, such as Gd2(L7)(H2O)2 were developed to ensure complete exclusion of the probe from intermolecular surfaces [117,165]. sPREs are measured as differences in relaxation rates (R1 or R2) or signal intensities of a biomolecule in the presence and absence of the paramagnetic probe, and can be obtained for any NMR-active nucleus (e.g. 1H, 13C, 15N, 31P) .
Solvent paramagnetic relaxation enhancement (sPRE) is a versatile nuclear magnetic resonance (NMR)-based method that allows characterization of the structure and dynamics of biomolecular systems through providing quantitative experimental information on solvent accessibility of NMR-active nuclei. Addition of soluble paramagnetic probes to the solution of a biomolecule leads to paramagnetic relaxation enhancement in a concentration-dependent manner. Here we review recent progress in the sPRE-based characterization of structural and dynamic properties of biomolecules and their complexes, and aim to deliver a comprehensive illustration of a growing number of applications of the method to various biological systems. We discuss the physical principles of sPRE measurements and provide an overview of available co-solute paramagnetic probes. We then explore how sPRE, in combination with complementary biophysical techniques, can further advance biomolecular structure determination, identification of interaction surfaces within protein complexes, and probing of conformational changes and low-population transient states, as well as deliver insights into weak, nonspecific, and transient interactions between proteins and co-solutes. In addition, we present examples of how the incorporation of solvent paramagnetic probes can improve the sensitivity of NMR experiments and discuss the prospects of applying sPRE to NMR metabolomics, drug discovery, and the study of intrinsically disordered proteins.
Probing Surfaces in Dynamic Protein Interactions
2020, Journal of Molecular Biology
Citation Excerpt :
Changes in the sPRE are compared and interpreted in terms of differential shielding of the spin of interest upon addition of the ligand, which allows for identification of changes in interaction sites. This approach has been successfully applied to a wide range of biomolecules, including proteins [92,93], nucleic acids , protein–protein , protein–nucleic acid , protein–membrane , protein–ligand, or RNA–ligand complexes . Here we focus on the application of the sPRE approach for the analysis of dynamic PPIs.
Proteins and their interactions control a plethora of biological functions and enable life. Protein–protein interactions can be highly dynamic, involve proteins with different degrees of “foldedness,” and are often regulated through an intricate network of post-translational modifications. Central parts of protein–protein networks are intrinsically disordered proteins (IDPs). IDPs act as regulatory interaction hubs, enabled by their flexible nature. They employ various modes of binding mechanisms, from folding upon ligand binding to formation of highly dynamic “fuzzy” protein–protein complexes. Mutations or perturbations in regulation of IDPs are hallmarks of many diseases. Protein surfaces play key roles in protein–protein interactions. However, protein surfaces and protein surface accessibility are difficult to study experimentally. NMR-based solvent paramagnetic relaxation enhancement (sPRE) can provide quantitative experimental information on protein surface accessibility, which can be further used to obtain distance information for structure determination, identification of interaction surfaces, conformational changes, and identification of low-populated transient structure and long-range contacts in IDPs and dynamic protein–protein interactions. In this review, we present and discuss state-of the art sPRE techniques and their applications to investigate structure and dynamics of IDPs and protein–protein interactions. Finally, we provide an outline for potential future applications of the sPRE approach in combination with complementary techniques and modeling, to study novel paradigms, such as liquid–liquid phase separation, regulation of IDPs and protein–protein interactions by post-translational modifications, and targeting of disordered proteins.
Investigations into the killing activity of an antimicrobial peptide active against extensively antibiotic-resistant K. pneumon iae and P. aeruginosa
2017, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
TOCSY (total correlation spectroscopy) spectra of the peptide-micelle system were acquired in the presence and absence of increasing concentrations of the paramagnetic probe Gd(III)(DTPA-BMA). The soluble probe causes nuclear spin relaxation proportional to the local surface accessibility of the molecule investigated [42–44]. Relaxation is measured by calculating the decrease in peak volumes on addition of the probe, and is summarized as a bare number, the attenuation value A, which ranges from 0 to 2 and can be determined for each known proton peak.
SET-M33 is a multimeric antimicrobial peptide active against Gram-negative bacteria in vitro and in vivo. Insights into its killing mechanism could elucidate correlations with selectivity.
SET-M33 showed concentration-dependent bactericidal activity against colistin-susceptible and resistant isolates of P. aeruginosa and K. pneumoniae. Scanning and transmission microscopy studies showed that SET-M33 generated cell blisters, blebs, membrane stacks and deep craters in K. pneumoniae and P. aeruginosa cells. NMR analysis and CD spectra in the presence of sodium dodecyl sulfate micelles showed a transition from an unstructured state to a stable α-helix, driving the peptide to arrange itself on the surface of micelles.
SET-M33 kills Gram-negative bacteria after an initial interaction with bacterial LPS. The molecule becomes then embedded in the outer membrane surface, thereby impairing cell function. This activity of SET-M33, in contrast to other similar antimicrobial peptides such as colistin, does not generate resistant mutants after 24h of exposure, non-specific interactions or toxicity against eukaryotic cell membranes, suggesting that SET-M33 is a promising new option for the treatment of Gram-negative antibiotic-resistant infections.
Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies
2014, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
MD data have been used to monitor the stability of intramolecular hydrogen bond, HB, networks [25,26] and the complete profiles of HB occurrences are shown in Fig. 2. As previously described [32–34], a good correlation between surface accessibility of protein HB donors and TEMPOL induced paramagnetic effects is observed for CXCL12. Backbone amides exhibiting Ai values higher than 1.6, indeed, have accessible surface areas, ASA, larger than 0 and, at the same time, low HB occupancies.
Hotspot delineation on protein surfaces represents a fundamental step for targeting protein–protein interfaces. Disruptors of protein–protein interactions can be designed provided that the sterical features of binding pockets, including the transient ones, can be defined. Molecular Dynamics, MD, simulations have been used as a reliable framework for identifying transient pocket openings on the protein surface. Accessible surface area and intramolecular H-bond involvement of protein backbone amides are proposed as descriptors for characterizing binding pocket occurrence and evolution along MD trajectories. TEMPOL induced paramagnetic perturbations on 1H–15N HSQC signals of protein backbone amides have been analyzed as a fragment-based search for surface hotspots, in order to validate MD predicted pockets. This procedure has been applied to CXCL12, a small chemokine responsible for tumor progression and proliferation. From combined analysis of MD data and paramagnetic profiles, two CXCL12 sites suitable for the binding of small molecules were identified. One of these sites is the already well characterized CXCL12 region involved in the binding to CXCR4 receptor. The other one is a transient pocket predicted by Molecular Dynamics simulations, which could not be observed from static analysis of CXCL12 PDB structures. The present results indicate how TEMPOL, instrumental in identifying this transient pocket, can be a powerful tool to delineate minor conformations which can be highly relevant in dynamic discovery of antitumoral drugs.
2022, Chemical Reviews
2020, Biophysical Reviews
First in vivo MRI study on theranostic dendrimersomes
Journal of Controlled Release, Volume 248, 2017, pp. 45-52
Amphiphilic Janus-dendrimers are able to self-assemble into nanosized vesicles named dendrimersomes. We recently synthesized the 3,5-C12-EG-(OH)4 dendrimer that generates dendrimersomes with very promising safety and stability profiles, that can be loaded with different contrast agents for in vivo imaging. In this contribution, nanovesicles were loaded with both the Magnetic Resonance Imaging (MRI) reporter GdDOTAGA(C18)2 and the glucocorticoid drug Prednisolone Phosphate (PLP), in order to test their effective potential as theranostic nanocarriers on murine melanoma tumour models. The incorporation of GdDOTAGA(C18)2 into the membrane resulted in dendrimersomes with a high longitudinal relaxivity (r1=39.1mM−1s−1, at 310K and 40MHz) so that, after intravenous administration, T1-weighted MRI showed a consistent contrast enhancement in the tumour area. Furthermore, the nanovesicles encapsulated PLP with good efficiency and displayed anti-tumour activity both in vitro and in vivo, thus enabling their practical use for biomedical theranostic applications.
The amyloid precursor protein (APP) intracellular domain regulates translation of p44, a short isoform of p53, through an IRES-dependent mechanism
Neurobiology of Aging, Volume 36, Issue 10, 2015, pp. 2725-2736
p44 is a short isoform of the tumor suppressor protein p53 that is regulated in an age-dependent manner. When overexpressed in the mouse, it causes a progeroid phenotype that includes premature cognitive decline, synaptic defects, and hyperphosphorylation of tau. The hyperphosphorylation of tau has recently been linked to the ability of p44 to regulate transcription of relevant tau kinases. Here, we report that the amyloid precursor protein (APP) intracellular domain (AICD), which results from the processing of the APP, regulates translation of p44 through a cap-independent mechanism that requires direct binding to the second internal ribosome entry site (IRES) of the p53 mRNA. We also report that AICD associates with nucleolin, an already known IRES-specific trans-acting factor that binds with p53 IRES elements and regulates translation of p53 isoforms. The potential biological impact of our findings was assessed in a mouse model of Alzheimer's disease. In conclusion, our study reveals a novel aspect of AICD and p53/p44 biology and provides a possible molecular link between APP, p44, and tau.
Efficient digestion of chitosan using chitosanase immobilized on silica-gel for the production of multisize chitooligosaccharides
Process Biochemistry, Volume 49, Issue 12, 2014, pp. 2107-2113
Chitosanase-coated silica-gels were prepared via cross-linking of the chitosanase onto silica-gels for the efficient production of multisize chitooligosaccharides (MCOs) in a continuous process. The kinetic aspects of immobilized chitosanase (IMMCTase) were investigated based on the reaction time, production of MCOs, and MALDI-TOF mass analyses to achieve maximum bioconversion of high molecular weight chitosan (HMWC) to MCOs. IMMCTase revealed a negligible loss of chitosanase activity after multi uses in continuous digestion of HMWC. The optimal temperature of IMMCTase was 37°C, and kinetic parameters toward HMWC were determined to be Km=1.45mM and Vmax=360μmole/μg/min, respectively. Under optimal conditions, the recovery of enzyme activity of IMMCTase was determined to be 82.3%, thus indicating that it can still be reused few more times. In conclusion, use of IMMCTase resulted in rapid and efficient digestions of HMWC with consistent results to produce MCOs.
Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1)
Journal of Molecular Biology, Volume 430, Issue 9, 2018, pp. 1295-1310
Cytochrome P450cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1H–15N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed.
Non-Heme Iron-Dependent Enzymes That Cleave Carbon-Carbon Bonds During Phosphonate Biosynthesis
Comprehensive Natural Products III, Volume 5, 2020, pp. 173-190
Mononuclear non-heme iron-dependent (NHI) enzymes catalyze an array of chemical transformations including hydroxylation, halogenation, and epimerization as well as both cyclization and ring cleavage reactions. These transformations occur in primary and secondary metabolism. A subset of this group of enzymes activates O2 without requiring cosubstrates, cofactors, or reductants. These enzymes extract all four electrons from their organic substrates for complete reduction of oxygen. This article focuses on two enzymes involved in phosphonate biosynthesis that utilize a ferric-superoxo intermediate to initiate catalysis and that involve a subsequent ferryl intermediate for a second step of oxidation. Both enzymes catalyze carbon-carbon bond scission in the same substrate 2-hydroxyethyl phosphonate leading to two products, but the extents of oxidation of the two products is different. One enzyme, 2-hydroxyethylphosphonate dioxygenase, is part of the biosynthesis of the commercially used herbicide phosphinothricin, whereas the second enzyme, methyl phosphonate synthase, catalyzes an important step in the carbon and phosphorus cycles in the oceans. Studies have shown that these two enzymes have a common mechanism up to a branch point and that branching is controlled by a single second sphere amino acid. The article discusses the evolution of the mechanistic and structural understanding of these enzymes.
Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides
Biophysical Chemistry, Volume 196, 2015, pp. 40-52
Plasticins (PTC) are dermaseptin-related antimicrobial peptides characterized by a large number of leucine and glycine residues arranged in GXXXG motifs that are often described to promote helix association within biological membranes. We report the structure and interaction properties of two plasticins, PTC-B1 from Phyllomedusa bicolor and a cationic analog of PTC-DA1 from Pachymedusa dacnicolor, which exhibit membrane-lytic activities on a broad range of microorganisms. Despite a high number of glycine, CD and NMR spectroscopy show that the two plasticins adopt mainly alpha-helical conformations in a wide variety of environments such as trifluoroethanol, detergent micelles and lipid vesicles. In DPC and SDS, plasticins adopt well-defined helices that lie parallel to the micelle surface, all glycine residues being located on the solvent-exposed face. Spectroscopic data and cross-linking experiments indicate that the GXXXG repeats in these amphipathic helices do not provide a strong oligomerization interface, suggesting a different role from GXXXG motifs found in transmembrane helices.
Copyright © 2012 Elsevier Inc. All rights reserved.