FTIR Analysis of Protein Structure

Samples were filtered with a 0.1 μm filter (Ultrafree-MC Centrifugal Filter, Merck, Darmstadt, Germany, 0.1 μm PVDF). The retentate was washed on the filter with a 50 mM deuterated glycine buffer pD 1.5. Both fractions as well as the entire solution mixture were vacuum dried at 43 °C for 6 h and resuspended in 50 mM deuterated glycine buffer at pD 1.5. FTIR measurements were performed on the retentate, the permeate, and the total fraction immediately after dissolution or washing. The measurements were performed on a Cary 630 FTIR spectroscope (Agilent Technologies, Santa Clara, CA, USA). The laser power was set to a gain of 2000 and a wavelength range of 2000 to 900 cm⁻¹ was scanned with a resolution of 0.25 cm⁻¹. Every spectrum was collected as an average of ten scans. For the structural analysis, the amide I region (1700 to 1600 cm⁻¹) was deconvoluted into Voigt peaks [41 (link)] at the maxima within the region 1616–1627 cm⁻¹ for the parallel β-sheets, 1628–1634 cm⁻¹ for antiparallel β-sheets, 1643–1647 cm⁻¹ for unordered part, 1650–1657 cm⁻¹ for α-helix and 1660–1675 cm⁻¹ for turns and loops [42 (link),43 (link),44 (link)].