Structural dynamics of the Ca2+-ATPase of sarcoplasmic reticulum. Temperature profiles of fluorescence polarization and intramolecular energy transfer

Istvan Jona, Janos Matko, Anthony Martonosi

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Abstract

The temperature dependence of fluorescence polarization and Förster-type resonance energy transfer (FRET) was analyzed in the Ca2+-ATPase of sarcoplasmic reticulum using protein tryptophan and site-specific fluorescence indicators such as 5-[2-((iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (IAEDANS), fluorescein 5′-isothiocyanate (FITC), 2′,3′-O-(2,4,3-trinitrophenyl)adenosine monophosphate (TNP-AMP) or lanthanides (Pr3+, Nd3+) as probes. The normalized energy transfer efficiency between AEDANS bound at cysteine-670 and -674 and FITC bound at lysine-515 increases with increasing temperature in the range of 10-37°C, indicating the existence of a relatively flexible structure in the region of the ATPase molecule that links the AEDANS to the FITC site. These observations are consistent with the theory of Somogyi, Matko, Papp, Hevessy, Welch and Damjanovich (Biochemistry 23 (1984) 3403-3411) that thermally induced structural fluctuations increase the energy transfer. Structural fluctuations were also evident in the energy transfer between FITC linked to the nucleotide-binding domain and Nd3+ bound at the putative Ca2+ sites. By contrast the normalized energy transfer efficiency between AEDANS and Pr3+ was relatively insensitive to temperature, suggesting that the region between cysteine-670 and the putative Ca2+ site monitored by the AEDANS-Pr3+ pair is relatively rigid. A combination of the energy transfer data with the structural information derived from analysis of Ca2+-ATPase crystals yields a structural model, in which the location of the AEDANS-, FITC- and Ca2+ sites are tentatively identified.

Original languageEnglish
Pages (from-to)183-199
Number of pages17
JournalBBA - Biomembranes
Volume1028
Issue number2
DOIs
Publication statusPublished - Oct 5 1990

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Keywords

  • (Sarcoplasmic reticulum)
  • ATPase
  • Ca-
  • Calcium ion transport
  • Fluorescence energy transfer
  • Molecular dynamics

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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