Fourier transform infrared spectroscopic studies of the secondary structure of spectrin under different ionic strengths

Abstract

Spectrin, a highly dynamic skeletal membrane protein, plays an important role in maintaining the disk biconcave shape of the human erythrocyte. The sequence of spectrin is mostly composed of repeating segments of 106 amino acids which have been proposed to form unique structural domains. Electronic and vibrational circular dichroism and Fourier transform infrared (FTIR) spectroscopy were used as complementary techniques to study the secondary structure of spectrin. The amide I and II regions of the FTIR absorbance spectra were analyzed using partial least-squares analysis. The secondary structure of spectrin under physiological buffer conditions was estimated to be about 70% alpha-helix, 10% beta-sheet, and 20% other. We believe that this is the first detailed experimental evidence of significant beta-sheet content in spectrin secondary structure. The antiparallel beta-sheet SH3 domain in the center of the alpha-subunit in spectrin accounts for only about 1.5% of the total amino acid residues in the dimer. Hydrodynamic studies have shown spectrin to be sensitive to changes in ionic strength and to addition of denaturing agents. Our FTIR results showed that the secondary structure of spectrin treated with detergent or NaOH changed by 10-20%. The Stokes radii of the spectrin samples used for FTIR measurements were found to vary as a function of the ionic strength, but their secondary structures did not change as a function of ionic strength. These results indicate that while the overall hydrodynamic dimension of spectrin depends on the medium ionic strength, the secondary structure remains essentially constant.(ABSTRACT TRUNCATED AT 250 WORDS)