Epitope Mapping of Ligand−Receptor Interactions by Diffusion NMR

Yan, Jiangli; Kline, Allen D.; Mo, Huaping; Zartler, Edward R.; Shapiro, Michael J.

doi:10.1021/ja0264347

Cited by 53 publications

(55 citation statements)

References 20 publications

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“…[39][40][41] The translational diffusion coefficient (D) is related to the mass of a complex through hydrodynamic radius. We used this technique to investigate the aggregation of the wild-type and G169D mutant Nramp1-TM4 in 40% HFIP-d 2 aqueous solution.…”

Section: Self-assemblymentioning

confidence: 99%

HFIP‐induced structures and assemblies of the peptides from the transmembrane domain 4 of membrane protein Nramp1

Xue

Wang

et al. 2006

Biopolymers

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Membrane protein Nramp1 (natural resistance-associated macrophage protein 1) is a pH-dependent divalent metal cation transporter that regulates macrophage activation in infectious and autoimmune diseases. A naturally occurring glycine to aspartic acid substitution at position 169 (G169D) within the transmembrane domain 4 (TM4) of Nramp1 makes mice susceptible to Leishmania donovani, Salmonella typhimurium, and Mycobacterium bovis. Here we present a structural and self-assembling study on two synthetic 24-residue peptides, corresponding to TM4 of mouse Nramp1 and its G169D mutant, respectively, in 1,1,1,3,3,3-hexafluoroisopropanol-d(2) (HFIP-d(2)) aqueous solution by nuclear magnetic resonance (NMR) spectroscopy. The results show that amphipathic alpha-helical structures are formed from residue Ile173 to Tyr187 for the wild-type peptide and from Trp168 to Tyr187 for the G169D mutant, respectively. The segment of the N-terminus from Leu167 to Leu172 is poorly structured for the wild-type peptide, whereas it is well defined for the G169D mutant. Both peptides aggregate to form a tetramer and the monomeric peptides in peptide bundles are structurally and orientationally similar. The intermolecular interactions in assemblies could be stronger in the C-terminal regions related to residues Phe180-Leu184 than those in the central helical segments for both peptides. The G169D mutation may change the size of the opening on the termini of assembly.

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Section: Self-assemblymentioning

confidence: 99%

HFIP‐induced structures and assemblies of the peptides from the transmembrane domain 4 of membrane protein Nramp1

Xue

Wang

et al. 2006

Biopolymers

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“…1 A host of NMR methods have been developed to screen for weak ligand-protein interactions in solution and to measure binding constants. Most methods exploit the various NMR properties of the ligand that are modulated by its association with the protein, and include saturation transfer, 2 measurements of diffusion by gradient spectroscopy, 3 transferred NOE experiments 4 and chemical shift mapping. 5 Such methods rely on the fact that, when the ligand is present in large excess over the protein, the spectrum of the ligand in solution carries information about its affinity for the protein, which can usually be extracted by one-or two-parameter fitting procedures.…”

Section: Introductionmentioning

confidence: 99%

Insights into the interactions between a drug and a membrane protein target by fluorine cross‐polarization magic angle spinning NMR

Boland

Middleton

2004

Magnetic Reson in Chemistry

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The fluorinated anti-psychotic drug trifluoperazine (TFP) has been shown to be a K(+)-competitive inhibitor of gastric H(+)/K(+)-ATPase, a membrane-embedded therapeutic target for peptic ulcer disease. This paper describes how variable contact time (19)F cross-polarization magic angle spinning (VCT-CP/MAS) NMR has been used to probe the inhibitory interactions between TFP and H(+)/K(+)-ATPase in native gastric membranes. The (19)F CP/MAS spectra for TFP in H(+)/K(+)-ATPase enriched (GI) gastric membranes and in control membranes containing less than 5 nmol of the protein indicated that the drug associates with the membranes independently of the presence of H(+)/K(+)-ATPase. The (19)F peak intensities in the VCT-CP/MAS experiment confirmed that TFP undergoes slow dissociation (k(off) < 100 s(-1)) from binding sites in GI membranes, and more rapid dissociation (k(off) < 100 s(-1)) from control membranes. The spectra showed that up to 40% of bound TFP was displaced from GI membranes by 100 mM K(+) and by the K(+)-competitive inhibitor TMPIP, but TFP was not displaced from the control membranes. Hence the spectra of TFP in GI membranes represent the drug bound to the K(+)-competitive inhibitory site of H(+)/K(+)-ATPase and to other non-specific sites. The affinity of TFP for the K(+)-competitive site (K(D) = 4 mM) was determined from a binding curve of (19)F peak intensity versus TFP concentration after correction for non-specific binding. The K(D) was much higher than the IC(50) for ATPase inhibition (8 microM), which suggests that the substantial non-specific binding of TFP to the membranes contributes to ATPase inhibition. This novel approach to probing ligand binding can be applied to a wide range of membrane-embedded pharmaceutical targets, such as G-protein coupled receptors and ion channels, regardless of the size of the protein or strength of binding.

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“…Recent results have shown that when the protein background is suppressed in a CPMG-filtered diffusion experiment, curved diffusion data (indicating trNOE) is still observed in instances of strong ligand-protein binding (ϳM K d ), but not significantly where binding is weak (ϳmM K d ) (84,85). In these systems, chemical exchange was fast on both the chemical shift and diffusion time scales; however, a slower off-rate likely facilitates observation of the trNOE at shorter ⌬ because binding is stronger.…”

Section: Applications Of Pfg-nmr Diffusion Measurements For Charactermentioning

confidence: 99%

“…Differential trNOE effects on a single ligand can potentially reveal which functional groups interact specifically with the protein, facilitating the analysis of ligand structure-activity relationships. Such trNOE detected in diffusion measurements has been quantified and validated against other NMR methods for determining ligand conformation (85), offering a faster and simpler approach for these measurements compared with 2D NMR techniques.…”

Section: Applications Of Pfg-nmr Diffusion Measurements For Charactermentioning

confidence: 99%

Measuring ligand‐protein binding using NMR diffusion experiments

Lucas

Larive

2004

Concepts Magnetic Resonance

104

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ABSTRACT:Characterization of ligand-protein interactions is important because many biologically important processes are mediated by the binding of a small molecule to an enzyme or cell-surface receptor. Pulsed-field gradient nuclear magnetic resonance (PFG-NMR) spectroscopy measurements of diffusion coefficients permit noninvasive, quantitative analysis of binding over a broad range of dissociation constants and ligand:protein concentration ratios. An arsenal of specific and selective PFG-NMR methods has been developed by exploiting differential behaviors of small molecule ligands and macromolecular proteins. A discussion of several PFG-NMR methods and their relevant applications reveals the analytical value of using PFG-NMR diffusion measurements for studying ligandprotein binding.

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Epitope Mapping of Ligand−Receptor Interactions by Diffusion NMR

Cited by 53 publications

References 20 publications

HFIP‐induced structures and assemblies of the peptides from the transmembrane domain 4 of membrane protein Nramp1

HFIP‐induced structures and assemblies of the peptides from the transmembrane domain 4 of membrane protein Nramp1

Insights into the interactions between a drug and a membrane protein target by fluorine cross‐polarization magic angle spinning NMR

Measuring ligand‐protein binding using NMR diffusion experiments

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