title: The nucleotide-binding site of the sarcoplasmic reticulum Ca-ATPase is conformationally altered in aged skeletal muscle.
abstract: 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.
authors.affiliation: Department of Molecular Biosciences, Biochemistry and Biophysics Section, Haworth Hall, University of Kansas, Lawrence, KS 66045-2106, USA.
authors.forename: B
authors.initials: B
authors.lastname: Chen
authors.forename: T E
authors.initials: TE
authors.lastname: Jones
authors.forename: D J
authors.initials: DJ
authors.lastname: Bigelow
mesh_terms: 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
chemical_list: D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D009711:Nucleotides; D012997:Solvents; C006421:1,5-I-AEDANS; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate