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 re combinant human factor XIII in aqueous solutions were investigated usi ng infrared and circular dichroism (CD) spectroscopies. The infrared a mide I spectrum of the protein in H2O solution at 25 degrees C exhibit ed an absorbance maximum near 1642 cm(-1), indicating the presence of a predominantly beta-sheet structure. Quantitative analysis revealed t hat the native protein contains 13-16% alpha-helix, 41-49% beta-sheet, 29% beta-turn, and 10-14% extended strand structures. The presence of a strong Low wavenumber beta-sheet band at 1641 cm(-1) and a weak hig h-wavenumber beta-sheet band at 1689 cm(-1) indicated that the beta-sh eet structure of the protein is predominantly antiparallel. Quantitati ve analysis of the CD spectrum using the SELCON method indicated a sec ondary structural content of 10% alpha-helix, 40-50% beta-sheet, 20-35 % beta-turns, and 20-35% unordered elements, which matches that determ ined by X-ray crystallography. The apparent discrepancy with the conte nts of unordered element determined by infrared spectroscopy is reconc iled by considering that CD spectroscopy and X-ray crystallography ass ign extended loops and strands to unordered elements, whereas infrared spectroscopy recognizes these as distinct structured elements. During heating above 60 degrees C, a pair of new infrared bands appeared at 1626 and 1693 cm(-1) for the protein in H2O and 1619 and 1683 cm(-1) i n D2O, indicating a formation of intermolecular beta-sheet aggregates. The intensities of the new bands increased as a function of temperatu re, concomitant with an intensity decrease in bands for the native pro tein structural elements. As expected, there was an increase in therma l stability in D2O relative to that in H2O, which was manifested as an increase of about 5 degrees C in the temperature for initial loss of infrared bands assigned to native structural elements and for appearan ce of bands due to intermolecular beta-sheet. In addition, the midpoin t of the thermally induced transitions in infrared spectra were about 2.5 degrees C higher in D2O than in H2O. Based on the infrared analysi s, the thermally denatured state of the protein in both H2O and D2O co ntains predominantly intermolecular beta-sheet. The broad, poorly reso lved absorbance that spans the region between the intermolecular beta- sheet bands was assigned to an ensemble of heterogeneous structural el ements (including unordered), none of which is populated to a high eno ugh degree to result in a distinct infrared band. Results from CD spec troscopy support these conclusions about the structure of the denature d, aggregated protein. (C) 1997 Academic Press.