Back to our protein whose possible identity is CTP synthetase, translation is needed in order to obtain the single letter code for the amino acids that our protein code for. An online translate tool is used (see: http://web.expasy.org/translate/).
With the single letter code obtained for our protein sequence, we are able to obtain its molecular weight and isoelectric point via a MW/Pi computational tool (see: http://web.expasy.org/compute_pi/).
The molecular weight of our protein is 38586.03, which is about 38.6kDa and the isoelectric point of our protein is 5.67. Pi is the pH where our protein does not carry any charge. The Pi of the protein will determine the charge of the protein. When the pH is lower than its Pi, the protein will carry a net positive charge and vice versa. This means that at pH above 5.67, the protein will be negatively charged. By know the Pi value, we can manipulate the pH and obtain the protein in a particular charge that we want. Thus, Pi value is useful as it enables us to separate proteins by isoelectric focusing, which uses a pH gradient to separate proteins. The Pi value of a protein also affects its solubility, in which at its Pi value, the protein will have minimal solubility and hence will precipitate. This is also what allows the separation of proteins; at its Pi, the protein will precipitate and no longer migrate towards the anode/cathode.
In order to find out the localization of our protein, we use a program called PSORT (see: http://www.psort.org/psortb/index.html). This program allows the prediction localization site of interested proteins by searching for amino acid sequences that match known localization signals. Some localization signals include ER signal consisting of KDEL and peroxisome signal consisting of SKL. Note that it is required to input what type of organism our sequence is from as localization signals differ greatly from different origin.
From the results of PSORT, it is predicted our protein can be found in the cytosolic membrane. By cross referencing on PubMed and other databases, the biological function of our predicted protein, CTP synthetase can be determined.
CTP synthetase is an enzyme in charge of catalysing the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP), a substrate needed for RNA synthesis. Due to this function, CTP synthetase would normally be localized in the cytosol. However, our results from psort predicted that it is localized in the cytoplasmic membrane instead. This is highly possible due recent findings that CTP synthetase has a second function of forming cytoskeletal filaments1. Cytoskeletal proteins are highly important not just for a cell’s structure and integrity, but also transport and cell division. In this case, CTP synthetase is involved in formation of filaments for the cytoplasmic membrane.
Due to its involvement with formation of filaments, CTP synthetase is co-localized with the filament it forms, polymerizing into it and into the cytoplasmic membrane. This justifies the psort localization prediction. As filaments are involved in the determination of cell shape, a study by Ingerson-Mahar et al. has been done to show that CTP synthetase negatively regulates the curvature of a cell. This means that cells depleted of CTP synthetase will be more curved than usual. However, the cell shape seems to be dependent on other factors and not just CTP synthetase as it also forms filaments in cells that are not curved.
It has also been shown that CTP synthetase filament forming function is independent of its enzymatic ability2, meaning it is bifunctional and is capable of performing one function without another. In fact, CTP synthetase is found to be incapable of enzymatic activity once it is involved in polymerization of filaments. However, this is reversible as the enzymatic activity of CTP synthetase is rendered inactive only till its dissociation from the polymer of filament.