Analysis of the structural organization and thermal stability of two spermadhesins. Calorimetric, circular dichroic and Fourier-transform infrared spectroscopic studies.
The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel beta-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP 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 Hcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that beta-structure and turns 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 that the porcine and the bovine proteins contain little, if any, alpha-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-beta-structure proteins, and would support a beta-barrel topology like that predicted for the CUB domain. Other beta-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques 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.
Menéndez
M
M
Instituto de Química-Física Rocasolano CSIC, Madrid, Spain.
Gasset
M
M
Laynez
J
J
López-Zumel
C
C
Usobiaga
P
P
Töpfer-Petersen
E
E
Calvete
J J
JJ
D000818:Animals; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D008297:Male; D029584:Prostatic Secretory Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012661:Semen / Q000737:chemistry*; D029589:Seminal Plasma Proteins; D017550:Spectroscopy, Fourier Transform Infrared; D013078:Sperm Immobilizing Agents / Q000737:chemistry; D013094:Spermatozoa / Q000737:chemistry*; D013552:Swine; D013696:Temperature
D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D029584:Prostatic Secretory Proteins; D011506:Proteins; D029589:Seminal Plasma Proteins; D013078:Sperm Immobilizing Agents; C043076:beta-microseminoprotein