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 [13C]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 [13C]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 α-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 st...