The structure of ovalbumin consists of four domains (Figure 3). Domains A, B and C contains both alpha helices and beta strands. Domain D is the smaller loop of mostly alpha helices.
Figure 3. The ovalbumin protein with the four domains labeled, A, B, C and D. The alpha helices and beta strands can be observed in this schematic diagram. The highlighted area is the D chain. This domain contains only alpha helices and is referred to as the exposed loop of ovalbumin.
Understanding the purpose of the ovalbumin protein in egg white is still undergoing tremendous research. It has been through the cleavage of ovalbumin by subtilisin that has contributed to what is known today about this protein (Figure 4). The cleavage form of ovalbumin by this enzyme is plakalbumin. It was observed long ago that this reaction resulted in limited proteolysis of ovalbumin and little global conformational change. Few changes in properties were also taken note.
Figure 4. Subtilisin is used in the cleavage of the ovalbumin protein to form plakalbumin, which is then used in research to understand the structure and function of ovalbumin. The mechanism that is known for this reaction has not been observed directly, but has been proven from studies with alpha-1-antitrypsin, a protein that is similar in reactive site to ovalbumin.
The exposed loop in ovalbumin, domain D (Figure 3), has been found to be inactive as opposed to other members of the serpin family that have been studied. This was proven with the H NMR spectrum of the different proteins of the serpin family in comparison with ovalbumin. Two other proteins which are used to study in order to understand the structure and function of ovalbumin are 1-alpha-antitrypsin and anthrombin III (Figures 8 and 9). Ovalbumin is one of the more conserved members of the serpin family, and shows a sequence identity of 30 percent in the tertiary structure of 1-alpha-antitrypsin. The proposed active site of ovalbumin, as found in 1-alpha-antitryspin, is expected to be at the Ala 358-Ser 359 center (Figures 5 and 6). However, no measurable inhibitory activities were observed and no conformational change could be found in ovalbumin. Whether with the exposed loop in its native form or cleaved from ovalbumin, the protein continues to remain stable in conformation and serves only as a protease substrate. Similar heat stabilities were also observed in the native and cleaved forms of ovalbumin. Therefore, ovalbumin has been used as a model for studies related to serpin family members because of its intact native form upon reaction and relative stability of the overall structure.
Figure 5. The proposed reactive center, Ser 359, is shown on the right of this diagram in CPK mode. This is the relaxed form of alpha-1-antitrypsin, which is the form resultant of cleavage of the active site, Met 358-Ser359. Methionine 358 is also in CPK mode on the bottome left corner of this protein. Observations based on the sequence alignment of this structure leads scientists to believe that the cleavage of ovalbumin by subtilisin occurs in a similar site at Ala 358-Ser 359.
Figure 6. This diagram shows ovalbumin with its reactive center site, Ala 358-Ser 359, which is located in Chain A of ovalbumin. The four domains of the protein can be observed in this model, with Chain A on the bottom left hand corner. The reactive site is located near the center of the ovalbumin protein, highlighted in yellow.
Some more detailed studies of the ovalbumin protein involving S-R conformational changes and phosphorylation of specific residues on the exposed loop. Many studies have proposed that native serpins exist in strained conformations which resulted in large conformational changes upon proteolysis to a relaxed form. Of course, ovalbumin continues to provide contradictory results.
Phosphorylation or removal of the Serine 344 group on the exposed loop has shown a failure of ovalbumin in producing conformation changes of S-R configuration. The suggested explanation of one research is that this observation is due to the unique presense of the bulky side chains at the P10-P12 site. This site is found to be well conserved in the members of the serpin group, excepting ovalbumin and another protein, angiotensinogen. The residues found at P11 and P12 in ovalbumin are both valine and those found in angiotensinogen are a proline and glutamate, respectively. These residues are larger than those found in the serpins studied, which contained mostly small residues of alanine (Figure 7). For S-R conformational changes observed in cleaving exposed loops found on other native serpin structures, a folding of the A4 strand into the major pleated sheet is required. This is due to the tight pocket formed by the phenylalanine residues and methionine residue (374). Because of the large residues found in ovalbumin, the movement of the A4 strand is prevented and no changes in S-R conformation is observed.
Figure 7. The two residues at sites P11 and P12 are highlighted in yellow, which are Val 79 and Val 80, respectively. Due to the large residues, the two valine groups inhibit ovalbumin from undergoing an R-S conformational arrangement upon cleavage of the exposed loop from the ovalbumin structure shown here. The peptide numbering is based on the sequence of the alpha-1-antitrypsin molecule.
As a result, phosphate groups on the exposed loop do not contribute to the failure of the conformational changes in ovalbumin. The variation in specific residues of the exposed loop affect the cleavaging of ovalbumin and its stability in its native structure. This leads to the inactive function of ovalbumin as an inhibitory protein. Instead, ovalbumin remains as a protease substrate upon reaction with other enzymes and cleavage of various sites.