ANALYSIS OF THE STRUCTURAL ORGANIZATION AND THERMAL-STABILITY OF 2 SPERMADHESINS - CALORIMETRIC, CIRCULAR DICHROIC AND FOURIER-TRANSFORM INFRARED SPECTROSCOPIC STUDIES

The CUB domain is a widespread 110-amino-acid module found in function ally diverse, often developmentally regulated proteins, for which an a ntiparallel beta-barrel topology similar to that in immunoglobulin V d omains has been predicted. Spermadhesins have been proposed as a subgr oup of this protein family built up by a single CUB domain architectur e. To test the proposed structural model, we have analyzed the structu ral organization of two members of the spermadhesin protein family, po rcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovi ne acidic seminal fluid protein (aSFP) homodimer, using differential s canning calorimetry, far-ultraviolet circular dichroism and Fourier-tr ansform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and a SFP were irreversible and followed a one step process with transition temperatures (Tm) of 60,5 degrees C and 78.6 degrees C, respectively. The calorimetric enthalpy changes (Delta H-cal) of thermal denaturatio n were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Anal ysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform i nfrared spectroscopy and deconvolution of circular dichroic spectra us ing a convex constraint analysis indicated that beta-structure and tur ns are the major structural element of both PSP-I/PSP-II (53% of beta- sheet, 21% of turns) and aSFP (44% of beta-sheet, 36% of turns), and t hat the porcine and the bovine proteins contain little, if any, alpha- helical structure. Taken together, our results indicate that the porci ne and the bovine spermadhesin molecules are probably all-beta-structu re proteins, and would support a beta-barrel topology like that predic ted for the CUB domain. Other beta-structure folds, such as the Creek- key pattern characteristic of many carbohydrate-binding protein domain s cannot be eliminated. Finally, the same combination of biophysical t echniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.