The inhibitory action of phospholamban involves stabilization of alpha-helices within the Ca-ATPase.
We have used attenuated total reflection Fourier transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopies to identify secondary and dynamic structural changes within the Ca-ATPase that result from the functional inhibition of transport activity by phospholamban (PLB). Isotopically labeled [(13)C]PLB was expressed and purified from Escherichia coli and was functionally reconstituted with unlabeled Ca-ATPase, permitting the resolution of the amide I and II absorbance bands of the Ca-ATPase from those of [(13)C]PLB. Upon co-reconstitution of the Ca-ATPase with PLB, spectral shifts are observed in both the CD spectra and the amide I and II bands associated with the Ca-ATPase, which are indicative of increased alpha-helical stability. Corresponding changes in the kinetics of H/D exchange occur upon association with PLB, indicating that 100 +/- 20 residues in the Ca-ATPase that normally undergo rapid amide H/D exchange become exchange resistant. There are no corresponding large changes in the secondary structure of PLB. The affinity of the structural interaction between PLB and the Ca-ATPase is virtually identical to that associated with functional inhibition (K(d) = 140 +/- 30 microM), confirming that the inhibitory regulation of the Ca-ATPase by PLB involves the stabilization of alpha-helices within the Ca-ATPase.
Tatulian
Suren A
SA
Biochemistry and Biophysics Section, Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045-2106, USA. statulia@mail.ucf.edu
Chen
Baowei
B
Li
Jinhui
J
Negash
Sewite
S
Middaugh
C Russell
CR
Bigelow
Diana J
DJ
Squier
Thomas C
TC
D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000099211:Phospholamban