Detection and Characterization of Boric Acid and Borate Ion Binding to Cytochrome c Using Multiple Quantum Filtered NMR

Taler, Galia; Eliav, Uzi

doi:10.1006/jmre.1999.1853

Cited by 14 publications

(10 citation statements)

References 19 publications

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“…The presence of these groups on proteins suggests that analogous protein complexes should exist. Consistent with this expectation, binding interactions between borate and cytochrome c have been detected by NMR spectroscopy (8,9). Noncovalently associated boric acid (10; pdb code 1B33) and borate (11; pdb code 1A95) occasionally have been observed in protein crystal structures; however, we are not aware of previous reports of covalent complexes involving borate and the residues on protein surfaces.…”

Section: Introductionsupporting

confidence: 65%

“…Since protein surfaces contain the same functional groups present in many small borate ligands, it was hypothesized that low-affinity borate binding sites might be a general characteristic of protein surfaces. NMR evidence for the existence of such sites in cytochrome c has previously been reported (8,9). Exploration of the surface of the trypsin molecule immediately revealed a serine-rich region on the surface including residues Ser164, Ser166, Ser167, Ser170, and Ser171 as a potential borate binding site.…”

Section: Nmr Studies Of the Trypsin-borate Complexmentioning

confidence: 87%

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NMR and Crystallographic Characterization of Adventitious Borate Binding by Trypsin

2006

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Recent 11B NMR studies of the formation of ternary complexes of trypsin, borate, and S1-binding alcohols revealed evidence for an additional binding interaction external to the enzyme active site. We have explored this binding interaction as a prototypical interaction of borate and boronate ligands with residues on the protein surface. NMR studies of trypsin in which the active site is blocked with leupeptin or with the irreversible inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) indicate the existence of a low-affinity borate binding site with an apparent dissociation constant of 97 mM, measured at pH 8.0. Observation of a field-dependent dynamic frequency shift of the (11)B resonance indicates that it corresponds to a complex for which omegatau >> 1. The 0.12 ppm shift difference of the borate resonances measured at 11.75 and 7.05 T, corresponds to a quadrupole coupling constant of 260 kHz. A much larger 2.0 ppm shift is observed in the 11B NMR spectra of trypsin complexed with benzene boronic acid (BBA), leading to a calculated quadrupole coupling constant of 1.1 MHz for this complex. Crystallographic studies identify the second borate binding site as a serine-rich region on the surface of the molecule. Specifically, a complex obtained at pH 10.6 shows a borate ion covalently bonded to the hydroxyl oxygen atoms of Ser164 and Ser167, with additional stabilization coming from two hydrogen-bonding interactions. A similar structure, although with low occupancy (30%), is observed for a trypsin-BBA complex. In this case, the BBA is also observed in the active site, covalently bound in two different conformations to both His57 Nepsilon and Ser195 Ogamma. An analysis of pairwise hydroxyl oxygen distances was able to predict the secondary borate binding site in porcine trypsin, and this approach is potentially useful for prediction of borate binding sites on the surfaces of other proteins. However, the distances between the Ser164/Ser167 Ogamma atoms in all of the reported trypsin crystal structures is significantly greater than the Ogamma distances of 2.2 and 1.9 angstroms observed in the trypsin complexes with borate and BBA, respectively. Thus, the ability of the hydroxyl oxygens to adopt a sufficiently close orientation to allow bidentate ligation is a critical limit on the borate binding affinity of surface-accessible serine/threonine/tyrosine residues.

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Section: Introductionsupporting

confidence: 65%

Section: Nmr Studies Of the Trypsin-borate Complexmentioning

confidence: 87%

NMR and Crystallographic Characterization of Adventitious Borate Binding by Trypsin

2006

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“…The putative lysine-based complexation occurring in this system that underlies the gel stiffening transition is not unprecedented. In fact, studies in the literature have focused on the complexation and hydrogen-bonding capability of boric acid/borate ion and boronic acid with lysine residues or other amine containing amino acids for a variety of biological systems. − ,, For example, Taler et al investigated the hydrogen bonding of borate ion with lysine using 1 H and 11 B NMR as a model system for studying the binding of phosphate ions to cytochrome c, a protein that has many lysine residues on its surface. − Their studies showed that borate ion formed hydrogen bonds with the lysine amine nitrogen and also formed salt bridges between other residues. In addition to biological systems, others have shown that the hydrogen-bonding capability of boric acid with amines, such as melamine, can be used in supramolecular chemistry .…”

Section: Resultsmentioning

confidence: 99%

Reversible Stiffening Transition in β-Hairpin Hydrogels Induced by Ion Complexation

et al. 2007

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We have previously shown that properly designed lysine and valine-rich peptides undergo a random coil to beta-hairpin transition followed by intermolecular self-assembly into a fibrillar hydrogel network only after the peptide solutions are heated above the intramolecular folding transition temperature. Here we report that these hydrogels also undergo a stiffening transition as they are cooled below a critical temperature only when boric acid is used to buffer the peptide solution. This stiffening transition is characterized by rheology, dynamic light scattering, and small angle neutron scattering. Rheological measurements show that the stiffening transition causes an increase in the hydrogel storage modulus (G') by as much as 1 order of magnitude and is completely reversible on subsequently raising the temperature. Although this reversible transition exhibits rheological properties that are similar to polyol/borax solutions, the underlying mechanism does not involve hydroxyl-borate complexation. The stiffening transition is mainly caused by the interactions between lysine and boric acid/borate anion and is not driven by the changes in the secondary structure of the beta-hairpin peptide. Addition of glucose to boric acid and peptide solution disrupts the stiffening transition due to competitive glucose-borate complexation.

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“…There was some interest in using boron containing substances as analogues for naturally occurring molecules, applying 11 B NMR to study their binding properties to biological macromolecules. [2][3][4] However, the majority of publications in this field and devoted to investigations related to molecules used in an experimental cancer treatment known as boron neutron capture therapy (BNCT), and these will be the main subject of the present review.…”

Section: Introductionmentioning

confidence: 99%

Biomedical applications of10B and11B NMR

Bendel

2005

NMR Biomed.

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This review focuses mainly on the detection and investigation of molecules used for boron neutron capture therapy (BNCT) by 10 B and 11 B NMR. In this binary radiation treatment, boron-containing molecules (also called 'BNCT agents') enriched in the 10 B isotope, are targeted to the tumor, and irradiated with thermal or epithermal neutrons. Capture of these neutrons by 10 B nuclei generates cell-damaging radiation, confined to single cell dimensions. NMR research efforts have primarily been applied in two directions: first, to investigate the metabolism and pharmaco-kinetics of BNCT agents invivo, and second, to use localized NMR spectroscopy and/or MRI for non-invasive mapping of the administered molecules in treated animals or patients. While the first goal can be pursued using 11 B NMR for natural-abundance samples (80% 11 B / 20% 10 B), molecules used in the actual treatment are >95% enriched in 10 B, and must therefore be detected by 10 B NMR. Both 10 B (spin 3) and 11 B (spin 3/2) are quadrupolar nuclei, and their typical relaxation times, in common BNCT agents in biological environments, are rather short. This poses some technical challenges, particularly for MRI, which will be reviewed, along with possible solutions. The first attempts at 11 B NMR and MRI detection of BNCT agents in biological tissue were conducted over a decade ago. Since then, results from 11 B MRI in laboratory animals and in humans have been reported, and 11 B NMR spectroscopy provided interesting and unique information about the metabolism of some BNCT agents in cultured cells. 10 B NMR was applied either 'indirectly' (in double-resonance experiments involving coupled protons), but also by direct 10 B MRI in mice. However, no results involving the NMR detection of 10 B-enriched compounds in treated patients have been reported yet.

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Detection and Characterization of Boric Acid and Borate Ion Binding to Cytochrome c Using Multiple Quantum Filtered NMR

Cited by 14 publications

References 19 publications

NMR and Crystallographic Characterization of Adventitious Borate Binding by Trypsin

NMR and Crystallographic Characterization of Adventitious Borate Binding by Trypsin

Reversible Stiffening Transition in β-Hairpin Hydrogels Induced by Ion Complexation

Biomedical applications of10B and11B NMR

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