CHARACTERIZATION OF VITELLINE MEMBRANE OUTER LAYER PROTEIN-I, VMO-I -AMINO-ACID-SEQUENCE AND STRUCTURAL STABILITY

Vitelline membrane outer layer protein I (VMO-I) tightly bound to ovom ucin fibrils of hen's egg yolk membrane was characterized in terms of its amino acid sequence and structural stability. The deduced sequence of VMO-I using the conventional sequencing method is: VLVGDDLSWGRFGPW SKRCKICGLQTKVESPQGLRDDTALNNVRFFCCK. Thus, VMO-I is composed of 163 ami no acid residues with a calculated molecular weight of 17,979. The seq uence confirms the cDNA sequence of VMO-I we recently determined and d oes not show any significant similarity to proteins compiled in the NB RF database. Two of the four disulfide bonds found in VMO-I were estim ated to lie between Cys26 and Cys57 and between Cys79 and Cys110. The sequence analyses show that VMO-I contains three 53-residue internal r epeats that contain distinctive regions of turns flanked by beta-sheet s consistent with the recent finding that the molecule contains a new beta-fold motif, the beta-prism. The molecular characteristics of VMO- I in solution were examined by CD spectroscopy in the far and near ult raviolet regions, NMR spectroscopy, and high sensitive differential sc anning calorimetry (DSC). CD spectra in the far UV region at room temp erature were similar to that assigned to a random coil, while in the n ear UV region, small positive peaks were observed. The ellipticity in both regions decreased on raising the temperature. Proton NMR experime nts showed the native structure unfolds to unordered conformations at 70 degrees C. A single positive peak appeared in the DSC curve with th e transition temperature of 67.5 degrees C, from which an unfolding en thalpy of 107 kcal/mol and a heat capacity change of 2.6 kcal/mol/K we re obtained. These results showed a normal degree of stability for the beta-prism structure found in VMO-I.