An NMR Investigation of the Conformational Effect of Nitroxide Spin Labels on Ala-Rich Helical Peptides

Bolin, Kimberly A.; Hanson, P. J.; Wright, Sarah; Millhauser, Glenn L.

doi:10.1006/jmre.1998.1365

Cited by 22 publications

(16 citation statements)

References 24 publications

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“…Magnetic dipole interactions between the unpaired electron of MTSL and nuclei that are within 13 Å of it result in the drastic suppression of their NMR signals. [29][30][31][32][33][34][35][36][37] The amino acids tryptophan and tyrosine are introduced as the 'fingerprint' functional groups in this method. Tryptophan and tyrosine have aromatic side chains, whose signals in 1D-proton NMR spectra are well-separated from all other proton signals, making the signal-quenching effect of the spin label unambiguous.…”

Section: Introductionmentioning

confidence: 99%

Probing coiled-coil assembly by paramagnetic NMR spectroscopy

et al. 2015

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Here a new method to determine the oligomeric state and orientation of coiled-coil peptide motifs is described. Peptides K and E, which are designed to form a parallel heterodimeric complex in aqueous solution, were labeled with the aromatic amino acids tryptophan and tyrosine on the C-terminus respectively as 'fingerprint' residues. One of the peptides was also labeled with the paramagnetic probe MTSL. One dimensional proton NMR spectroscopy was used to study the peptide quaternary structure by monitoring the signal suppression of the aromatic labels due to proximity of the nitroxyl radical. 1D-NMR confirmed that the peptides K and E form a heterodimeric coiled coil with a parallel orientation. In addition, fluorescence emission quenching of the aromatic labels due to electron exchange with a nitroxyl radical confirmed the parallel coiled coil orientation. Thus, paramagnetic nitroxide and aromatic fluorophore labeling of peptides yields valuable information regarding the quaternary structure from 1D-NMR and steady-state fluorescence measurements. This convenient method is useful not only to investigate coiled coil assembly, but can also be applied to any defined supramolecular assembly.

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Section: Introductionmentioning

confidence: 99%

Probing coiled-coil assembly by paramagnetic NMR spectroscopy

et al. 2015

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“…Even at buried sites, no significant perturbation of the backbone is evident (19,45). Finally, others have reported modest or negligible effects of spin labeling on protein folding (47) or backbone structure of peptides as measured by NMR (48). This can be attributed to the relatively compact size of the modified Cys residue (a molecular volume on the order of Tyr) and its ambivalent chemical nature, which does not favor highly polar or nonpolar environments.…”

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confidence: 99%

Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1

Nygaard

Lagerstedt

Bjerre

et al. 2011

Journal of Biological Chemistry

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We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na The ubiquitous plasma membrane Na ϩ /H ϩ exchanger isoform 1 (NHE1) 4 plays central roles in cellular pH and volume homeostasis, cell migration, proliferation, and survival, and increased NHE1 activity contributes to ischemia-reperfusion injury as well as tumor growth and proliferation (1, 2). Hence, the ability to selectively block NHE1, although of high clinical relevance, is hampered by a lack of detailed understanding of NHE1 structure and mechanism of ion translocation. Hydropathy analyses and accessibility studies indicate that NHE1 has 12 transmembrane (TM) segments and a large Cterminal cytoplasmic region (3). Cysteine accessibility studies suggest the presence of two small re-entrant loops between TM IV and TM V (intracellular loop (IL) II) and TM VIII and TM IX (IL IV), respectively, and a larger re-entrant loop between TM IX and TM X (extracellular loop V) (1, 3). Portions of IL II and IL IV are located within the membrane and accessible from either side of the membrane, suggesting that they may form structures lining an aqueous pore and could be involved in ion translocation by NHE1 (3). Extracellular loop V is also interesting in this regard, because it resembles the Ploops found in voltage-gated ion channels (1, 3). These putative re-entrant loops are highly conserved among several NHE1 homologs, consistent with the notion that they are critical for NHE1 function (3-5).A number of regions within the NHE1 protein have been implicated in inhibitor binding, e.g. TM IV and TM IX (6 -11); however, the mechanism(s) of interaction between NHE1 and its commonly used inhibitors, amiloride and benzoyl guanidine type compounds, remain to be fully elucidated.Using a comparative approach based on chimeras generated using human NHE1 (hNHE1) and two NHE1 homologs (flounder paNHE1 and Amphiuma tridactylum NHE1) with high sequence homology to hNHE1 yet markedly different inhibitor profiles (4, 5), we previously obtained novel information on the regions of NHE1 important for inhibitor binding and ion transport (12). These studies confirmed that TM IV plays a central role in inhibitor binding (12) as suggested by earlier point mutation studies (6 -11). Moreover, we demonstrated that regions in TM X-XI and/or IL V and extracellular loop VI are important determinants of inhibitor sensitivity (12).The three-dimensional structure of NHE1 is unknown; however, the structure of the distantly related bacterial (Esch-

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“…A conformational shift (experimental chemical shift minus random coil chemical shift) of −0.1 ppm indicates helix and a conformational shift of 0.1 ppm indicates β‐strand. We have shown previously that gentle ascorbic acid reduction of the nitroxide to the hydroxylamine allows for detailed NMR analysis of spin‐labeled peptides without interference from the paramagnetic center [19]. Using two‐dimensional NOESY and TOCSY, the conformational shifts were determined for all three peptides and the results are compared to that of the original 3K sequence in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Methods for determining two‐dimensional NMR spectra of spin‐labeled peptides have been described previously [19]. Briefly, all NMR spectra were recorded on a Varian Unity+ 500 MHz spectrometer.…”

Section: Methodsmentioning

confidence: 99%

Dap‐SL: a new site‐directed nitroxide spin labeling approach for determining structure and motions in synthesized peptides and proteins

et al. 2002

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A new approach for site-directed placement of nitroxide spin labels in chemically synthesized peptides and proteins is described. The scheme takes advantage of a novel diaminopropionic acid sca¡old to independently control backbone and side chain elongation. The result is a spin-labeled side chain, referred to as Dap-SL, in which an amide bond forms a linker between the nitroxide and the peptide backbone. The method was demonstrated in a series of helical peptides. Circular dichroism and nuclear magnetic resonance showed that Dap-SL introduces only a minor perturbation in the helical structure. The electron paramagnetic resonance spectrum of the singly labeled species allowed for determination of the spin label rotational correlation time and suggests that the Dap-SL side chain is more £exible than the modi¢ed Cys side chain frequently used in site-directed spin label studies. Spectra of the doubly labeled peptides indicate a mixture of 3 10 -helix and K K-helix, which parallels ¢ndings from previous studies. The scheme demonstrated here o¡ers a fundamentally new approach for introducing spin labels into proteins and promises to signi¢cantly extend biophysical investigations of large proteins and receptors. In addition, the technique is readily modi¢ed for incorporation of any biophysical probe. ß

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An NMR Investigation of the Conformational Effect of Nitroxide Spin Labels on Ala-Rich Helical Peptides

Cited by 22 publications

References 24 publications

Probing coiled-coil assembly by paramagnetic NMR spectroscopy

Probing coiled-coil assembly by paramagnetic NMR spectroscopy

Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1

Dap‐SL: a new site‐directed nitroxide spin labeling approach for determining structure and motions in synthesized peptides and proteins

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