Bw. Chen et al., The nucleotide-binding site of the sacroplasmic reticulum Ca-ATPase is conformationally altered in aged skeletal muscle, BIOCHEM, 38(45), 1999, pp. 14887-14896
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.