Team:KCL UK/Protein Engineering
Structural Modelling
Introduction
Here, we explain the processes and the development of the different protein engineering applications we decided complete with Pvfp-5ß to improve our proteins ability to function as a bio-adhesive in spinal cord injury applications. You can check out our other proposed solutions here (link this part) PROPOSED IMPLEMENTATION.
OXIDATION OF DOPA RESIDUES
Why is auto-oxidation so problematic?
In nature, MFPs are co-secreted alongside hydrogen ions which reduces the pH of the mussel foot; this is an adaptation that preserves the chemical structure of the DOPA residues by reducing the likelihood of DOPA oxidation, thus allowing the formation of coordinate bonds and hydrogen bonds. Exposure of Mfp-5 to a neutral pH results in >95% decrease in adhesion due to the oxidation of DOPA to DOPA-quinone, resulting in a loss of coordinate and hydrogen bonds. (1) This is problematic for our project, as implanting an MFP-based bioadhesive coated scaffold into the human body at physiological pH will result in the rapid oxidation of DOPA and a loss of adhesiveness of the bioadhesive polymer. Furthermore, DOPA-quinone encourages cross-linking with other DOPA residues and forms covalent bonds with nucleophilic groups, including -NH2, -SH and imidazole, found on biological substrates - an increase in cross linking results in reduced protein flexibility, a fundamental component of pvfp5ß adhesion. In oxidative conditions, reactive oxygen species can form, which can have detrimental effects, leading to a cascade of damage in the site of injury. (2) We concluded that the next stage of our research should be looking into protecting DOPA residues via synthetic and biomimetic methods detailed below.
Despite efforts to prevent oxidation of DOPA, it is a fundamental part of the formation of the mussel byssus, factoring towards the structural integrity of the byssal thread. This occurs through DOPA-quinone, the oxidised form of DOPA, which can polymerise into chains that the mussel uses to form the mussel byssus. This is shown in figure X below. During the formation of the mussel byssus, Fe3+ ions and O2 leak into the mussel foot, initiating the oxidative process and beginning crosslinking. Nearing the end of the formation of the byssal thread, the mussel foot is lifted, leading to an influx of pH 8 seawater and metal ions. This leads to mass oxidation across the byssal thread, solidifying, strengthening and giving it its supporting shape, forming the intermediate between the adhesive surface and the mussel.
I am a placeholder for an Figure X.
We understood that we would need to balance the amounts of DOPA and DOPA-quinone in order to balance the adhesiveness and the structure of our protein polymer, and we optimised an in vivo method to do this, shown below:
PROTEIN STABILITY
here we can write about iGAM
POLYMERISATION
this will be all about tyrosinase, we can link throughly to the PARTS section
PROTEIN MODIFICATION
speak about the RGD peptide, and using the EDC/NHS coupling mechanism to link PCL to MFP
MUTAGENESIS STUDIES
we can conclude all our iGAM research here, saving the other modelling/simulations we have done for the "model" section of the wiki
