Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban.

Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban. Research Abstract Details 

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Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban. Abstract Text:

Zhiwen Zhang,Henriette A Remmer,David D Thomas,Christine B Karim,

Electron paramagnetic resonance (EPR) was used to optimize the solid-phase peptide synthesis of a membrane-bound peptide labeled with TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid). The incorporation of this paramagnetic amino acid results in a nitroxide spin label coupled rigidly to the alpha-carbon, providing direct detection of peptide backbone dynamics by EPR. We applied this approach to phospholamban, which regulates cardiac calcium transport. The synthesis of this amphipathic 52-amino-acid membrane peptide including TOAC is a challenge, especially in the addition of TOAC and the next several amino acids. Therefore, EPR of synthetic intermediates, reconstituted into lipid bilayers, was used to ensure complete coupling and 9-fluorenylmethoxycarbonyl (Fmoc) deprotection. The attachment of Fmoc-TOAC-OH leads to strong immobilization of the spin label, whereas Fmoc deprotection dramatically mobilizes it, producing an EPR spectral peak that is completely resolved from that observed before deprotection. Similarly, coupling of the next amino acid (Ser) restores the spin label to strong immobilization, giving a peak that is completely resolved from that of the preceding step. For several subsequent steps, the effect of coupling and deprotection is similar but less dramatic. Thus, the sensitivity and resolution of EPR provides a quantitative monitor of completion at each of these critical steps in peptide synthesis. Mass spectrometry, circular dichroism, and Edman degradation were used in concert with EPR to verify the chemistry and characterize the secondary structure. In conclusion, the application of conventional analytical methods in combination with EPR offers an improved approach to optimize the accurate synthesis of TOAC spin-labeled membrane peptides.

Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban. Publishing Authors By Initials

Z Zhang,HA Remmer,DD Thomas,CB Karim,

For similar natural sciences: physics: thermodynamics research abstracts see: natural sciences: physics: thermodynamics research

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Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Biopolymers

VOLUME: 88

Page Numbers: 29-35

Journal Abbreviation: Biopolymers

ISSN: 0006-3525

DAY: 3

MONTH: 12

YEAR: 2007

Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban. Information

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LANGUAGE: eng

NlmUniqueID: 372525

Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban. Keywords Mesh Terms:

KEYWORDS: Thermodynamics

MESH TERMS: chemistry

Chemical & Substance for Abstract: Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban. Information

Substance Name: 2,2,6,6-tetramethylpiperidine-N-oxide-4-

Registry Number: 15871-57-5

Grant and Affiliation Information for Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban.

AFFILIATION: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

Country: United States

AGENCY: United States NIGMS

GRANT: GM27906

ACRONYM: GM

MEDLINETA: Biopolymers

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