Identification of CDeT11–24 Interaction Partners

4.4. Identification of CDeT11–24 Interaction

approach relies on the immunoadsorption of the pre-formed complexes on a column linked with the purified IgGs specific for the bait protein CDeT11–24. One reason for the failure of the coimmunoaffinity chromatography could be the binding strength of the purified IgGs bound to the column. The range of measured values of affinity constants for antibody–

antigen binding spans from 10⁵ M⁻¹ to 10¹² M⁻¹, whereas a typical enzyme–substrate interaction (e.g. trypsin) has an affinity constant of10⁴ M⁻¹ (Harlow and Lane,1988). In the case of a coiled-coil interaction, a typical binding constant for leucine zipper moieties that interact is in the range of10⁷ M⁻¹ (Phizicky and Fields, 1995). This points out the far greater affinity the IgGs have over other protein-protein interactions. Since they can differ in their affinity constants for several orders of magnitude, it is not unlikely that the interacting proteins could be displaced by the antibody molecules.

4.4.2. The Weak Affinity Chromatography Suggests that CDeT11–24 Interacts With Itself

To overcome the plausible drawback of the coimmunoaffinity chromatography another approach was taken, where the column was directly coupled with the bait protein. To do this two columns were prepared, one coupled with the unphosphorylated form of CDeT11–24, and a second one coupled with the phosphorylated CDeT11–24. For this pourpose the unphosphorylated protein consisted in the recombinant full length histidine-tagged CDeT11–24. For the phosphorylated form, the native CDeT11–24 protein was isolated from dried C. plantagineum leaves. In this way two different columns covalently linked to the two isoforms of the bait protein were made available. The matrix of the two columns differed in the phosphorylation status of the bait protein, thus enabling an affinity chromatography to enrich the plant extracts in proteins that were differentially interacting with the solid phase, depending on the secondary modification status of CDeT11–24.

The eluted fractions separated by SDS–PAGE indicate that the column coupled with the recombinant protein is able to retain different polypeptides, whereas the phosphorylated version of CDeT11–24 does not. Interestingly, the major band in the Coomassie-stained gel in Figure3.43has a molecular size compatible with that of the CDeT11–24 protein. Its identity was confirmed by Western blot analysis, indicating that the band corresponds to CDeT11–24 and besides the full length protein, other smaller bands are recognized by the antibody. The meaning of this remains elusive, perhaps indicating degradation products

of the protein. The Western blot analysis also revealed that from the column coupled with the native phosphorylated protein it was eluted a band corresponding to the CDeT11–24 protein. Since the protein could not be observed in the Coomassie-stained gel, it is likely that this weak signal comes from protein bleeding from the column.

The only sample showing potential interaction candidates is the one from the partially dried leaves loaded on the column coupled with the recombinant protein. This finding is in line with previous experiments conducted on CDeT11–24 by 2D Blue Native SDS–PAGE (H. Röhrig, personal communication). It was observed that in native conditions the 11–24 protein from partially dehydrated plants localizes in a region of the gel between 100 and 200 kilodaltons, whereas the protein from fully dehydrated plants occurs at its predicted monomeric molecular size.

The competition experiment depicted in Figure3.43 is aimed at confirming the specificity of the interaction between the bait protein and the potential interaction partners. An excess of the free CDeT11–24 protein added to the plant extract before the weak affin-ity chromatography would prevent the adsorption of the proteins on the column. The disappearance of bands in the lane treated with free bait protein would strengthen the assumption that they are specific interactors. The Coomassie-stained gel in Figure 3.43 reveals two bands showing the expected pattern. In order to identify them, the same samples were separated by 2D SDS–PAGE and the spots corresponding to the two can-didates subjected to MS/MS analysis. The Mascot search revealed that they correspond to CDeT11–24 fragments, indicating that the outcompeted proteins belong to the protein itself.

Nevertheless, the 2D SDS–PAGE revealed another intriguing aspect of the competition experiment. The full-size CDeT11–24 protein appears to localize at two different isoelec-tric points along the pH gradient in the first dimension. The spot in the gel without the competitor has a more acidic pI as compared to the same spot belonging to the sample with competitor. The two spots were analyzed by MS/MS revealing that in the sample with the free 11–24 protein added as competitor, peptides corresponding to the recombi-nant histidine-tagged protein were present. This finding is in accordance with the protein sequence of the recombinant protein, which is predicted to have a pI of 5.57 due to the basic histidine tag, whereas the native CDeT11–24 has a more acidic pI (4.73). The oc-currence of the histidine-tagged recombinant protein in the spot corresponding to the full length CDeT11–24 indicates that the protein is able to interact with itself in the

unphos-phorylated form, and this interaction can be depleted by adding an excess of recombinant 11–24, which outcompetes the binding of the native protein.

These results deliver additional informations about the features of the LEA-like protein CDeT11–24 ofC. plantagineum, indicating that phosphorylation is able to regulate protein oligomerization. LEA proteins have the capability to form homo-oligomers (Ceccardiet al., 1994;Kazuoka and Oeda,1994). It might be speculated that CDeT11–24, similar to what Goyal et al. (2003) propose for the nematode protein AavLEA1, could form higher order coiled-coil interactions with itself. These interactions could form a network of protein filaments reminiscent of the cytoskeletal intermediate filaments, thus providing additional mechanical support to the cell.

Alternatively, the phosphorylation-dependent release of the monomer in the later stages of dehydration could represent a mechanism of regulation of the protein based on the early production of CDeT11–24 when the plant is sensing the stress, and the phosphorylation-driven activation and liberation of a putative active monomeric form in the later stages of desiccation. Supporting this model is the fact that the desiccation sensitive plantL. sub-racemosa presents a protein isoform with a reduced or completely absent phosphorylated isoform, as reported in section3.4.

However, it has to be pointed out that this experiment was focused on the identification of potential proteinaceous interaction partners. It cannot be excluded that other compounds interact with CDeT11–24. CDeT11–24 contains a K-segment reminiscent of that occurring in the group 2 LEA proteins. For some proteins of this group, an ion-binding activity has been demonstrated (Battagliaet al.,2008). The acidic dehydrin VCaB45 from celery and the Arabidopsis ERD14 possess calcium-binding properties, a feature that seems to be positively mediated by phosphorylation (Heyen et al., 2002; Alsheikh et al., 2003, 2005).

Furthermore, recently it has been shown that the K-segment of the maize dehydrin DHN1 is able to bind to anionic phospholipids vesicles, and that the segment adapts anα-helical conformation upon binding (Koaget al., 2009).

In conclusion the oligomerization of the LEA-like CDeT11–24 protein could represent a mechanism for its action, together with other not yet unveiled putative functions related to known properties typical of dehydrins, like binding to ions and phospholipidis. In the latter case different approaches have to be considered to test the hypothesis of non-proteinaceous binding partners.