Difference between revisions of "Part:BBa K3011012"

Latest revision as of 03:17, 22 October 2019

Metal ion Chelator

We used the cytoplasmic domain of Human Copper Transporter (hCtr1) as an analogue of the metal ion chelator. hCtr1 transports copper with high affinity in a time-dependent and saturable manner and is metal-specific. hCtr1-mediated copper transport is an energy-independent process and is stimulated by extracellular acidic pH and high K(+) concentrations. hCtr1 exists as a homomultimer at the plasma membrane in mammalian cells. The part that we are using for our experimental purposes consists of only two ion binding sites.

Proteomic quantification

The protein was inserted and expressed in the PEG expression vector. We confirmed the expression of the protein in the bacterial system by using an SDS PAGE electrophoresis.

SDS PAGE electrophoresis comparison of hCt cytoplasmic domain expression by transformed bacteria in LB media and DMEM. The last lane was loaded with the purified protein to check as a positive control.

Binding Affinity to Iron

To check the binding affinity of the Human copper transporter(hCt) cytoplasmic domain to free iron cations we used Isothermal titration calorimetry (ITC). It measures the heat absorbed or generated when ligand interacts with the protein and is used to determine the thermodynamic parameters of interactions in solution. It is most often used to study the binding of small molecules (such as inorganic ions ) to larger macromolecules (proteins.) In our case, it helped us to determine whether hCt is able to chelate free iron ions present in the surrounding environment.

ITC data for the buffer buffer interaction. The buffer used in this case was Tris(pH 8.0)

ITC data for the protein-ligand interaction. The protein being the hCtr1 and the ligand is Iron

From the graph obtained for the buffer-buffer interaction we can see that there is almost negligible energy change indicating the fact that there is no interaction within the different components of the buffer. On the other hand, for the energy graph obtained from the protein-ligand interaction, there is a considerable amount of energy change. Moreover, the thermodynamic parameter data (N1 and N2) given by the experiment suggests for every protein macromolecule there are two binding sites. This is in clear correlation with the expectations as there we are expressing only two of the homeodomains which in principle should have two binding domains.

Sequence and Features