Analysis of the Structural Organization and Thermal Stability of two Spermadhesins

Abstract

The CUB domain is a widespread 1 lO-amino-acid twdule lound in fiinclionally diverse, often developmentally regulated proteins, for which an antiparallel P-b:itrel topology similar to that in irriniunoflobulin V domains has been predicted. Spemxidhesins have becn proposed as a subgroup of this protein family built up by B single CUB domain architccturc. To tcst the proposed structural model, wc have analyzed the structural organkition o f two meinher.; of the spei-madhesin protein frimily, porcine seminal plasma proteins 1/11 (PSP-I/PSP-TI) heterodimer arid bovinc acidic seminal fluid protein (aSFP) horiiodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and ~;ourici-transforrn infrared spcctroscopy. Thermal unfolding of PSP-l/PSP-II and aSFP werc irreversible and follocvcd a onc-step process with transition temperatures (Tin) of 60.S"C and 78.6"C, rcspectively. The calorimctric cnthalpy changes (AH,,,,) of thermal denaturation werc 439 k.l/niol for PSP-I/PSP-I1 and 660 kJ/tnol for aSIT dimer. Analysis of the calorimetric curves of PSP-I/PSP-I1 showcd thut the cntire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using ii convex constraint analysis indicated that [I-structurc arid turns are the miijor slructural element of both PSP-UPSP-I1 (53% of p-sheet, 21 %I of turns) and aSFP (44% of /I-shcet, 36% of turns), ant1 that the porcine and the bovine proteins contain little, if any, Ix-helical slructurc. Taken together, OLIT rcsults indicate that the porcine and the bovine spermadhesin molecules are probably all-/j-struclure proteins, and would support a /$-barrel topology like thal predicted for the CUB domain. Othcr p-structurc folds, such as the Greek-key pattern characteristic of inany carbohydrate-binding protein dotnains ciinnot be eliminated. Finally, the saiiic combination of biophysical tcchniques was used to characterize the residual secondary slructurc of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphustze the aggregation tendency of these lorms.Keywords: scminal plasrna proteins; spermadhesins; CUB domain; conformational analysis; calorimetry.The early events of niamtrialian fertilization involvc a coinplcx series of highly orchestrated interactions between complementary molecules located on the surface of homologous gametes. In mammiils, sperm-egg recognition m d binding i s mediated hy spermatozoa-associated carbohydrate-binding proteins and saccharide chains of the oocyte's cxtracellular matrix called thc zona pcllucida (reviewed 1 1 1 ) . A variety of sperm proteins with zona-pellucida-binding capability, both integral plasrnii membrane and peripherally associated proteins, have becn idcntified in dilferent nummalian species (see [2] and refcrences therein). This suggests that fertilization is probably mediated by multiple zona pellucida spermatozoon-receptor systems, which collectively dictate the species specificity of gamete interaction.Cr,rr~.sl)rttrdmrp t...

Control of the Structural Stability of the Tubulin Dimer by One High Affinity Bound Magnesium Ion at Nucleotide N-site

“…Control experiments were run in parallel at 7 mM Mg 2ϩ in the initial equilibration buffer. Circular Dichroism-The far-UV CD spectra of tubulin (1-5 M, equilibrated in PEDTA with 1 mM nucleotide and a known amount of Mg 2ϩ ) were acquired in a JASCO J720 dichrograph equipped with a temperature regulated cell holder (1,35), with a 0.1-cm cell at 20 Ϯ 1°C. Thermal denaturation was monitored following the variation in ellipticity at 220 nm, using a temperature scan rate of 0.5°C⅐min…”

Conformational Features and Thermal Stability of Bovine Seminal Plasma Protein PDC‐109 Oligomers and Phosphorylcholine‐Bound Complexes

“…Circular dichroism spectroscopy. Circular dichroism spectra were recorded in a JASCO J-720 spectropolarimeter fitted with a thermostated cell holder and interfaced to a thermostatic bath as previously described (MenCndez et al, 1995). Farultraviolet and near-ultraviolet spectra were recorded in 0.1 -cm pathlength quartz cells at a protein concentration of about 0.15 mg/ml and 0.7 mg/ml, respectively.…”

Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture

“…asymmetric unit. Previous biophysical studies suggested that aSFP is a homodimeric protein (MenCndez et al, 1995). However, because the crystallographic data can not rule out the possibility that aSFP crystallized as a dimer with internal crystallographic symmetry, determination of the molecular mass of aSFP was performed by analytical ultracentrifugation.…”

“…Both subunits of the spermadhesin PSP-I/PSP-I1 heterodimer are glycoproteins and have basic isoelectric points (8.0-8.6), whereas aSFP is an acidic nonglycosylated protein (PI 4.8) (Einspanier et al, 1991). In addition, thermal unfolding of aSFP has a transition temperature that is 18°C higher than that of PSP-I/PSP-I1 (78.6"C and 60.5 "C, respectively), and the calorimetric enthalpy changes of thermal denaturation are 439 and 660 kJ/mol for PSP-I/PSP-I1 and aSFP, respectively (MenCndez et al, 1995). Comparison of the surface potentials and interior packings of PSP-I/PSP-I1 and aSFP may provide clues to interpret the different biophysical and ligandbinding characteristics of these two spermadhesin molecules.…”