The nucleotide-binding site of the sarcoplasmic 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-ATPase, we have utilized the fluorescence properties of protein-bound probes to assess both local and global structure. We find conformational changes that include an age-related decrease in the apparent binding affinity to high affinity calcium sites detected by fluorescence signals in both tryptophans within nearby membrane-spanning helices and fluorescein isothiocyanate (FITC) bound distally to Lys(515) within the nucleotide-binding site. In addition, a substantial (80%) age-related increase in the accessibility to soluble quenchers of fluorescence of FITC is observed without concomitant changes in bimolecular quenching constants (k(q)) for protein-bound IAEDANS, also within the nucleotide-binding domain, and tryptophans within the membrane. Using fluorescence resonance energy transfer to measure distances between IAEDANS and FITC across the nucleotide-binding domain, we find no significant age-related change in the mean donor-acceptor distance; however, significant increases are observed in the conformational heterogeneity of this domain, as assessed by the width at half-maximum (HW) of the distance distribution, increasing with age from 29.4 +/- 0.8 A to 42.5 +/- 1. 1 A. Circular dichroism indicates that the average secondary structure is unaltered with age. Thus, these data suggest tertiary structural alterations in specific regions around the nucleotide-binding site rather than global conformational changes.
Chen
B
B
Department of Molecular Biosciences, Biochemistry and Biophysics Section, Haworth Hall, University of Kansas, Lawrence, KS 66045-2106, USA.
Jones
T E
TE
Bigelow
D J
DJ
D000375:Aging; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002942:Circular Dichroism; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes / Q000378:metabolism; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D011916:Rats, Inbred F344; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012997:Solvents; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D009711:Nucleotides; D012997:Solvents; C006421:1,5-I-AEDANS; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate