Spectroscopic Study of Secondary Structure and Thermal Denaturation of Recombinant Human Factor XIII in Aqueous Solution

The secondary structure and thermal denaturation (in H2O vs D2O) of recombinant human factor XIII in aqueous solutions were investigated using infrared and circular dichroism (CD) spectroscopies. The infrared amide I spectrum of the protein in H2O solution at 25°C exhibited an absorbance maximum near 1642 cm−1, indicating the presence of a predominantly β-sheet structure. Quantitative analysis revealed that the native protein contains 13–16% α-helix, 41–49% β-sheet, 29% β-turn, and 10–14% extended strand structures. The presence of a strong low-wavenumber β-sheet band at 1641 cm−1and a weak high-wavenumber β-sheet band at 1689 cm−1indicated that the β-sheet structure of the protein is predominantly antiparallel. Quantitative analysis of the CD spectrum using the SELCON method indicated a secondary structural content of 10% α-helix, 40–50% β-sheet, 20–35% β-turns, and 20–35% unordered elements, which matches that determined by X-ray crystallography. The apparent discrepancy with the contents of unordered element determined by infrared spectroscopy is reconciled by considering that CD spectroscopy and X-ray crystallography assign extended loops and strands to unordered elements, whereas infrared spectroscopy recognizes these as distinct structured elements. During heating above 60°C, a pair of new infrared bands appeared at 1626 and 1693 cm−1for the protein in H2O and 1619 and 1683 cm−1in D2O, indicating a formation of intermolecular β-sheet aggregates. The intensities of the new bands increased as a function of temperature, concomitant with an intensity decrease in bands for the native protein structural elements. As expected, there was an increase in thermal stability in D2O relative to that in H2O, which was manifested as an increase of about 5°C in the temperature for initial loss of infrared bands assigned to native structural elements and for appearance of bands due to intermolecular β-sheet. In addition, the midpoint of the thermally induced transitions in infrared spectra were about 2.5°C higher in D2O than in H2O. Based on the infrared analysis, the thermally denatured state of the protein in both H2O and D2O contains predominantly intermolecular β-sheet. The broad, poorly resolved absorbance that spans the region between the intermolecular β-sheet bands was assigned to an ensemble of heterogeneous structural elements (including unordered), none of which is populated to a high enough degree to result in a distinct infrared band. Results from CD spectroscopy support these conclusions about the structure of the denatured, aggregated protein.