Structural descriptions of ligands in their binding site of integral membrane proteins at near physiological conditions using solid-state NMR

Watts, Anthony; Burnett, Ian J.; Glaubitz, Clemens; Gröbner, Gerhard; Middleton, David A.; Spooner, Paul J. R.; Williamson, Philip T. F.

doi:10.1007/s002490050187

Cited by 13 publications

(5 citation statements)

References 30 publications

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“…The experiments were performed on a small molecule. They can be translated directly to biologically interesting compounds like neurotransmitters (e.g., acetylcholine where preliminary measurements have been performed) and hormones whose structure could be determined bound to proteins . We also foresee that the method is applicable to larger molecules like peptides.…”

Section: Discussionmentioning

confidence: 99%

Determination of Internuclear Distances in Uniformly Labeled Molecules by Rotational-Resonance Solid-State NMR

et al. 2003

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Rotational-resonance magic-angle spinning NMR experiments are frequently used to measure dipolar couplings and to determine internuclear distances. So far most measurements were performed on samples containing isolated spin pairs. Thus, extensive structure elucidation, for example in biomolecules, requires the preparation of a whole set of doubly labeled samples. Here, we describe the analysis of the rotational-resonance polarization-exchange curves obtained from a single, uniformly labeled sample. It is shown experimentally that, at a magnetic field of 14.09 T, the rotational-resonance conditions in uniformly (13)C-labeled threonine are sufficiently narrow to permit the measurement of five distances between the four carbon spins with an accuracy of better than 10%. The polarization-exchange curves are analyzed using a modified two-spin model consisting of the two active spins. The modified model includes an additional offset in the final polarization, which comes from the coupling to the additional, passive, spins. The validity of this approach is experimentally verified for uniformly (13)C-labeled threonine. The broader applicability of such a model is demonstrated by numerical simulations which quantify the errors as a function of the most relevant parameters in the spin system.

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Section: Discussionmentioning

confidence: 99%

Determination of Internuclear Distances in Uniformly Labeled Molecules by Rotational-Resonance Solid-State NMR

et al. 2003

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“…The interaction of peripheral proteins with biological lipid membranes plays a key role in many cellular processes and is typically governed by electrostatic interactions between positively charged protein residues and negatively charged membrane surfaces. − Examples of electrostatically driven processes include the adsorption of antimicrobial peptides and proteins such as Src and MARCKS that display clusters of basic residues triggering electrostatic anchoring to a membrane surface. ,,, Even amyloidogenic systems such as the amyloid-β (Aβ) protein, can associate electrostatically, where the dramatic increase of its local membrane surface concentration triggers accelerated aggregation into toxic protofibrils, a process currently debated as a major neurotoxic pathway in Alzheimer's disease. ,, Initial electrostatic binding of proteins to their target membranes is not only controlled by the presence of charges at the protein surface, but crucially by the lipid composition. , As the huge variety of anionic lipids and their distribution pattern suggests, essential electrostatic interactions occur not only on the surface but also across the whole membrane interface. Therefore, not only is the net charge of the lipid headgroup important but also the residual charge distribution within a lipid headgroup structure, indicating the physiological significance of the internal charge location. ,, While the structural understanding of proteins at a molecular level has enhanced our understanding of how they achieve their membrane-bound state, detailed information about the significance of the charge distribution inside the membrane interface (as controlled by various specific lipids), is still quite restricted. ,,,,, …”

Section: Introductionmentioning

confidence: 99%

Molecular Insight into the Electrostatic Membrane Surface Potential by¹⁴N/³¹P MAS NMR Spectroscopy: Nociceptin−Lipid Association

2005

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Exploiting naturally abundant (14)N and (31)P nuclei by high-resolution MAS NMR (magic angle spinning nuclear magnetic resonance) provides a molecular view of the electrostatic potential present at the surface of biological model membranes, the electrostatic charge distribution across the membrane interface, and changes that occur upon peptide association. The spectral resolution in (31)P and (14)N MAS NMR spectra is sufficient to probe directly the negatively charged phosphate and positively charged choline segment of the electrostatic P(-)-O-CH(2)-CH(2)-N(+)(CH(3))(3) headgroup dipole of zwitterionic DMPC (dimyristoylphosphatidylcholine) in mixed-lipid systems. The isotropic shifts report on the size of the potential existing at the phosphate and ammonium group within the lipid headgroup while the chemical shielding anisotropy ((31)P) and anisotropic quadrupolar interaction ((14)N) characterize changes in headgroup orientation in response to surface potential. The (31)P/(14)N isotropic chemical shifts for DMPC show opposing systematic changes in response to changing membrane potential, reflecting the size of the electrostatic potential at opposing ends of the P(-)-N(+) dipole. The orientational response of the DMPC lipid headgroup to electrostatic surface variations is visible in the anisotropic features of (14)N and (31)P NMR spectra. These features are analyzed in terms of a modified "molecular voltmeter" model, with changes in dynamic averaging reflecting the tilt of the C(beta)-N(+)(CH)(3) choline and PO(4)(-) segment. These properties have been exploited to characterize the changes in surface potential upon the binding of nociceptin to negatively charged membranes, a process assumed to proceed its agonistic binding to its opoid G-protein coupled receptor.

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“…Now, drugs, solutes and neuromodulators are being described structurally and kinetically in fully competent integral membrane receptors, transporters and channels. In addition, peptide and protein structures are being resolved in model membranes (Opella 1997;Watts et al 1998), in advance of the crystal structures, should these ever be resolved for the membraneembedded forms.…”

Section: Discussionmentioning

confidence: 99%

Structural Resolution of Ligand–Receptor Interactions in Functional, Membrane-embedded Receptors and Proteins using Novel, Non-perturbing Solid-state NMR Methods

Watts¹

1999

Pharmacy and Pharmacology Communications

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Derning structural details for membrane‐embedded proteins is limited by the availability of two‐ or three‐dimensional crystals suitable for diffraction studies. This is even more difrcult when the structure of a ligand in its binding site is required because difference crystallography would be necessary, and with two‐dimensional crystals, extramembraneous protein detail is often missing and resolution is low in the direction of the membrane normal. Also, many bioactive ligands do not have any structured form in solution (solvents, detergents, etc.), but it is usually accepted that their target site does have structural requirements which derne efrcacy and potency. To address the question of ligand structure for bound ligands, novel solid‐state NMR techniques were used to derne molecular constraints at atomic resolution for ligands at their site of action, in fully functional, hydrated proteins under near physiological conditions (4°C or higher), and in a form often used for pharmacological and biochemical characterization. Using the solid‐state NMR approach, the structure of retinal has been resolved in the bacterial proton pump, bacteriorhodopsin, and in the 7‐TMD G‐coupled receptor, mammalian rhodopsin, without any knowledge of the protein structure itself. The rrst structural details of a widely used drug analogue, an imidazopyridine inhibitor of the gastric H+‐K+‐ ATPase, at its site of action, have been determined. Other examples for large integral proteins include the observation and kinetic description of solutes in the antimicrobial target sugar transporter proteins of bacteria, and the resolution of the chemistry of the binding site of acetylcholine when in the binding site of the nicotinic acetylcholine receptor. In addition, small peptide ion channels are amenable to study using solid‐state NMR methods, and the state of oligomerization, residues involved in the channel, can now be derned, with the potential for describing functionally signircant residues and thus aid inhibitor or modulator design. The information gained and methods developed for small amounts of protein (μmolnmol of binding sites), open the way to derning structural details of peptide hormones, flexible drugs and other ligands bound at their site of action in a functional system. The results therefore have relevance to the in‐vivo situation, and are of direct importance for the understanding of structural requirements for ligand‐activated signal transduction and cellular activity. In some cases, the residues involved in binding are also being resolved, thereby giving a complete picture of the vital and highly relevant ligand‐binding site structure and environment, in the absence of knowledge of the protein backbone or its structural arrangements.

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Structural descriptions of ligands in their binding site of integral membrane proteins at near physiological conditions using solid-state NMR

Cited by 13 publications

References 30 publications

Determination of Internuclear Distances in Uniformly Labeled Molecules by Rotational-Resonance Solid-State NMR

Determination of Internuclear Distances in Uniformly Labeled Molecules by Rotational-Resonance Solid-State NMR

Molecular Insight into the Electrostatic Membrane Surface Potential by¹⁴N/³¹P MAS NMR Spectroscopy: Nociceptin−Lipid Association

Structural Resolution of Ligand–Receptor Interactions in Functional, Membrane-embedded Receptors and Proteins using Novel, Non-perturbing Solid-state NMR Methods

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Structural descriptions of ligands in their binding site of integral membrane proteins at near physiological conditions using solid-state NMR

Cited by 13 publications

References 30 publications

Determination of Internuclear Distances in Uniformly Labeled Molecules by Rotational-Resonance Solid-State NMR

Determination of Internuclear Distances in Uniformly Labeled Molecules by Rotational-Resonance Solid-State NMR

Molecular Insight into the Electrostatic Membrane Surface Potential by14N/31P MAS NMR Spectroscopy: Nociceptin−Lipid Association

Structural Resolution of Ligand–Receptor Interactions in Functional, Membrane-embedded Receptors and Proteins using Novel, Non-perturbing Solid-state NMR Methods

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Molecular Insight into the Electrostatic Membrane Surface Potential by¹⁴N/³¹P MAS NMR Spectroscopy: Nociceptin−Lipid Association