Centrin is a low molecular mass (20 kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. Local and overall changes were investigated for interactions between cations and Chlamydomonas centrin using Fourier transform infrared (FT-IR) and circular dichroic (CD) spectroscopies. FT-IR spectral features studied included the amide I' band and the side-chain absorbances for aspartate residues located almost exclusively at the calcium-binding sites in the spectral region of 1700-1500 cm-1. The amide I′ band is exquisitely sensitive to changes in protein secondary structure and is observed to shift from 1626.5 to 1642.7 cm-1 in the presence and absence of calcium. These spectral bands are complex and were further studied using two-dimensional Fourier transform infrared (2D-FT-IR) correlation along with curve-fitting routines. Using these methods the secondary structure contributions were determined for holocentrin and apocentrin. The α-helical content in centrin was determined to be 60%-53% in the presence and absence of cations, respectively. Furthermore, the β-strand content was determined to be 12%-36%, while the random coil component remained almost constant at 7%-13.5% in the presence and absence of cations, respectively. Changes in the side-chain band are mostly due to the monodentate coordination of aspartate to the cation. A shift of ∼4 cm-1 (for the COO-antisymmetric stretch in Asp) from 1565 to 1569 cm-1 is observed for apocentrin and holocentrin, respectively. Thermal dependence revealed reversible conformational transition temperatures for apocentrin at 37 °C and holocentrin at 45 °C, suggesting greater stability for holocentrin.
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