Download MSc in Biochemistry, MSc in Biotechnology, MSc in Bioorganic

MSc in Biochemistry, MSc in Biotechnology, MSc in Bioorganic
Dissertation Project – 2nd Cycle
Student´s Name:
Student email address:
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Supervisor(s): Ana Cecília Afonso Roque & Olga Iranzo
Supervisor(s) email address: [email protected] [email protected]
Lab/Institution: DQ, FCT/UNL (PT) & Institut des Sciences Moléculaires de Marseille (France)
TITLE: DESIGN AND SYNTHESIS OF SMALL PROTEIN-BASED SCAFFOLDS FOR BIOENGINEERING
APPLICATIONS
BACKGROUND
The design of novel proteins with tailor-made roperties and functionalities is a growing research discipline
in the fields of chemistry, bioengineering and biomedicine. This is a multidisciplinary endeavor that
requires the expertise of scientists from different areas.
Chemists and bioengineers can play a critical role in this effort developing innovative computational and
chemical strategies to produce robust scaffolds where one can have a precise control over the location of
crucial elements, such as functional groups for target recognition or metal ion binding. Therefore, these
structures can render new, cheaper and more stable alternatives for therapeutic, chemical, biochemical
and biotechnological applications.
Under these grounds, the aim of this project is to explore the potential of the small WW and Zinc Finger
(ZF) domains as basic scaffolds to generate robust structures. Theywill be engineered to develop affinity
ligands for bioseparation of phosphoproteins and metallopeptidases with tailored-made catalytic
properties. These novel scaffolds will have high impact in the areas of biomedicine and biotechnology,
respectively.
OBJECTIVES
The specific goals of the project include:
1) “In silico” design of scaffolds to attain the target functionality
2) Biological production of protein scaffolds in recombinant hosts or production by solid-phase peptide
synthesis and subsequent characterization and immobilization
3) Confirmation of target functionality: phosphoproteins/peptides binding affinity and specificity for WW
domains and peptidase activity for ZF domains
4) Characterization and optimisation of the lead ligands (key structural changes on the lead ligands will be
considered for improved activity)
PROJECT DESCRIPTION
The project will involve:
Task 1. “In silico” rational redesign of the WW and ZF domains to obtain lead candidates with the target
functionality.naturally occurring domains of ZF proteins as well as the WW domains either free or in
complex with their target binding molecules will be used as starting scaffold in our protein rational design,
using the Protein Databank as our primary structural source.
Task 2. Synthesis and characterization of the selected domains. These scaffolds will be either synthesized
on an automated peptide synthesizer using standard Fmoc solid-phase chemistry or obtained by
Escherichia coli expression using synthetic genes. Crude products will be purified by reverse-phase HPLC
and further analyzed by mass spectroscopy (ESI/MALDI-TOF).
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MSc in Biochemistry, MSc in Biotechnology, MSc in Bioorganic
Dissertation Project – 2nd Cycle
The secondary structure content and thermodynamic stability of the different designed scaffolds will be
studied by circular dichroism (CD) spectroscopy and if deemed necessary, by NMR spectroscopy.
Task 3. Study of their functional properties. The functionalities of the different scaffolds
(phosphoproteins/peptides binding affinity/specificity and peptidase activity) will be studied both free and
immobilised on supports (e.g. agarose and magnetic particles). WW domains will be screened in a highthroughput format for binding to selected phosphorylated and non-phosphorylated peptides and proteins
and their binding capability will be determined. The best candidates will be also tested for the purification
of phosphorylated proteins in real mixture samples. Thetransition metal ions coordination properties of ZF
domains will be studied using different spectroscopic techniques [UV-Vis, EPR, CD, fluorescence and
NMR].. Peptidase activity will be screened in a high-throughput format using a broad range of
commercially available model substrates as well as fluorogenic peptide substrates specific for different
peptidases. The best systems will be then challenged with more complex substrates and peptidase activity
will be assessed and compared with that from conventional peptidases. Both teams will supervise these
catalytic studies.
Task 4. Structure activity relationships and lead optimization. For both systems, activity will be correlated
with structure and sequence to obtain a deeper understanding of key factors necessaries for better
molecular recognition, biophysical properties and peptidase activity. This crucial information will lead the
future optimization of the scaffolds. Additionally, different spectroscopic methodologies will be used to
characterize the interaction between the best lead scaffolds and the target substrates.
Task 5 - Thesis writing
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**NOTE
Travelling and accommodation costs to conduct the work at the University of Marseille, France, will be
covered by a research grant between the two Institutions.
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