Protein Structure and Metal Ion Interactions Transglutaminases

Transglutaminases are catalysts that are involved in the post-translational modification of proteins, forming isopeptide bonds at glutamine residues (Greenberg et al. 1991; Folk and Finnlayson 1997). The primary function of these enzymes appears to be in the stabilisation of tissues and, as such, their occurrence is widespread, being found in both extracellular and cellular fluids. There is evidence that transglutaminases may be bifunctional, also having capabilities to perform the role of G-proteins in cell signalling pathways (Monsonego et al. 1998; Nakoaka et al. 1994).

Factor XIII is one of the best characterised transglutaminases, being the last enzyme to be activated in the blood coagulation cascade, increasing the mechanical strength and chemical stability of blood clots. It is a tetramer consisting of two catalytic "a" subunits and two non-catalytic "b" subunits (which are thought to play a role in stabilising the "a" units). The protein-modifying activities of transglutaminases play a role in programmed cell death, cross-linking membrane and cytoskeletal proteins (Fesus et al. 1991; Siefring et al. 1978). This activity has been shown to be modulated by calcium and GTP (Achyuthan and Greenburg 1987; Bergamini et al. 1993). This modulation is thought to be related to putative conformational changes induced by the modulators. Indirect analysis using shallow-angle neutron scattering and shallow-angle x-ray scattering (Cassadio et al. 1999) and circular dichroism spectroscopy (Di Venere et al. 2000) have suggested that there is a significant increase of 0.8 nm in the gyration radius of the protein on binding calcium, suggesting a significant broadening of the protein structure on binding. Efforts to elucidate conformational changes directly from crystallographic data have not been successful as it is thought that the active form (with calcium bound) is constrained by the crystal lattice (Fox et al. 1999). Reports on optical measurements of transglutaminase on the addition of calcium have also been reported in which anomalies in the expected responses have been attributed to likely conformational changes in the protein on binding calcium (Gestwicki et al. 2001).

Using DPI it has been possible to measure the conformational changes associated with calcium binding directly. The experiment was carried out as follows. The protein was immobilised to an amine functionalised sensor surface via free external amine groups on the protein using BS3. A thickness increase of 5 nm was observed, which demonstrates that the disc-shaped molecule (approximately 15 nm in diameter and 5nm thick) has been immobilised face-parallel to the chip surface, as would be expected using this immobilisation method. Once the protein surface was created, it was challenged with different concentrations of calcium chloride and chloride mole equivalents of sodium chloride. As can be seen from Fig. 10, the protein layer undergoes substantial conformational changes in the presence of calcium that do not occur in the presence of sodium. It was also possible, having conducted the experiment at a range of concentrations, to calculate the affinity constant for the interaction. The affinity constant has been calculated to be 1.16 mM, which compares well with the range of literature values for the interaction (0.2-3.0 mM; Mottahedeh and Marsh 1998). This clearly suggests that the structural changes observed are directly related to the binding of calcium.

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