The nucleotide-binding site of the sacroplasmic reticulum Ca-ATPase is conformationally altered in aged skeletal muscle

Cellular conditions in senescent skeletal muscle have been shown to result in the loss of conformational stability of the sarcoplasmic reticulum (SR) Ca-ATPase. To identify underlying structural features of age-modified Ca-AT Pase, we have utilized the fluorescence properties of protein-bound probes to assess both local and global structure. We find conformational changes t hat include an age-related decrease in the apparent binding affinity to hig h affinity calcium sites detected by fluorescence signals in both tryptopha ns within nearby membrane-spanning helices and fluorescein isothiocyanate ( FITC) bound distally to Lys(515) within the nucleotide-binding site. In add ition, a substantial (80%) age-related increase in the accessibility to sol uble quenchers of fluorescence of FITC is observed without concomitant chan ges in bimolecular quenching constants (k(q)) for protein-bound IAEDANS, al so within the nucleotide-binding domain, and tryptophans within the membran e. Using fluorescence resonance energy transfer to measure :distances betwe en IAEDANS and FITC across the nucleotide-binding domain, we find no signif icant age-related change in the mean donor-acceptor distance; however, sign ificant-increases are observed in the conformational heterogeneity of this domain, as assessed by the width at half-maximum (HW) of the distance distr ibution, increasing with age from 29.4 +/- 0.8 Angstrom to 42.5 +/- 1.1 Ang strom. Circular dichroism indicates that the average secondary structure is unaltered with age. Thus, these data suggest tertiary structural alteratio ns in specific regions around the nucleotide-binding site rather than globa l conformational changes.