Nitroxide Side-Chain Dynamics in a Spin-Labeled Helix-Forming Peptide Revealed by High-Frequency (139.5-GHz) EPR Spectroscopy

M. Bennati; G. J. Gerfen; G. V. Martinez; R. G. Griffin; D. J. Singel; G. L. Millhauser

doi:10.1006/jmre.1999.1769

Nitroxide Side-Chain Dynamics in a Spin-Labeled Helix-Forming Peptide Revealed by High-Frequency (139.5-GHz) EPR Spectroscopy

M. Bennati, G. J. Gerfen, G. V. Martinez, R. G. Griffin, D. J. Singel, G. L. Millhauser

Physiology & Biophysics

Research output: Contribution to journal › Article › peer-review

17 Scopus citations

Abstract

High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence Ac-(AAAAK)₂CAAAKA-NH₂ 3K-11, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy τ_⊥/τ_∥ (≡ N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.

Original language	English (US)
Pages (from-to)	281-286
Number of pages	6
Journal	Journal of Magnetic Resonance
Volume	139
Issue number	2
DOIs	https://doi.org/10.1006/jmre.1999.1769
State	Published - Aug 1999

Keywords

Anisotropic motion
EPR
High frequency
Peptide
Side-chain dynamics

ASJC Scopus subject areas

Biophysics
Biochemistry
Nuclear and High Energy Physics
Condensed Matter Physics

Access to Document

10.1006/jmre.1999.1769

Cite this

@article{bfc8d84d58a04a70ae3961669e973b04,

title = "Nitroxide Side-Chain Dynamics in a Spin-Labeled Helix-Forming Peptide Revealed by High-Frequency (139.5-GHz) EPR Spectroscopy",

abstract = "High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence Ac-(AAAAK)2CAAAKA-NH2 3K-11, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy τ⊥/τ∥ (≡ N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.",

keywords = "Anisotropic motion, EPR, High frequency, Peptide, Side-chain dynamics",

author = "M. Bennati and Gerfen, {G. J.} and Martinez, {G. V.} and Griffin, {R. G.} and Singel, {D. J.} and Millhauser, {G. L.}",

note = "Funding Information: This work was supported by grants from the National Institutes of Health GM-46870 (G.L.M.), RR-00995 (R.G.G.), and GM-38352 (R.G.G. and D.J.S.) and by a grant from the National Science Foundation MCB 9408284 (G.L.M.). M. Bennati expresses her gratitude to the Deutsche Forschungsgemeinshaft for a postdoctoral grant. G.V.M. was supported by an NIH predoctoral fellowship (GM-16396) and G.J.G. was supported by an American Cancer Society postdoctoral fellowship (PF-3668).",

year = "1999",

month = aug,

doi = "10.1006/jmre.1999.1769",

language = "English (US)",

volume = "139",

pages = "281--286",

journal = "Journal of Magnetic Resonance",

issn = "1090-7807",

publisher = "Academic Press Inc.",

number = "2",

}

TY - JOUR

T1 - Nitroxide Side-Chain Dynamics in a Spin-Labeled Helix-Forming Peptide Revealed by High-Frequency (139.5-GHz) EPR Spectroscopy

AU - Bennati, M.

AU - Gerfen, G. J.

AU - Martinez, G. V.

AU - Griffin, R. G.

AU - Singel, D. J.

AU - Millhauser, G. L.

N1 - Funding Information: This work was supported by grants from the National Institutes of Health GM-46870 (G.L.M.), RR-00995 (R.G.G.), and GM-38352 (R.G.G. and D.J.S.) and by a grant from the National Science Foundation MCB 9408284 (G.L.M.). M. Bennati expresses her gratitude to the Deutsche Forschungsgemeinshaft for a postdoctoral grant. G.V.M. was supported by an NIH predoctoral fellowship (GM-16396) and G.J.G. was supported by an American Cancer Society postdoctoral fellowship (PF-3668).

PY - 1999/8

Y1 - 1999/8

N2 - High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence Ac-(AAAAK)2CAAAKA-NH2 3K-11, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy τ⊥/τ∥ (≡ N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.

AB - High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence Ac-(AAAAK)2CAAAKA-NH2 3K-11, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy τ⊥/τ∥ (≡ N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.

KW - Anisotropic motion

KW - EPR

KW - High frequency

KW - Peptide

KW - Side-chain dynamics

UR - http://www.scopus.com/inward/record.url?scp=0033170414&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033170414&partnerID=8YFLogxK

U2 - 10.1006/jmre.1999.1769

DO - 10.1006/jmre.1999.1769

M3 - Article

C2 - 10423365

AN - SCOPUS:0033170414

SN - 1090-7807

VL - 139

SP - 281

EP - 286

JO - Journal of Magnetic Resonance

JF - Journal of Magnetic Resonance

IS - 2

ER -

Nitroxide Side-Chain Dynamics in a Spin-Labeled Helix-Forming Peptide Revealed by High-Frequency (139.5-GHz) EPR Spectroscopy

Abstract

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this