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Theme 1
Project 1:
Supervisor:
Molecular Design
Redesigning Protein Interactions through Chemical Modification
of the Protein Surface
Dr. Peter B. Crowley, School of Chemistry
Project Summary: Protein-protein interactions are central to
organisation and function in the living cell. We are interested
in understanding the determinants of protein recognition and
binding affinity. The aim of this project is to investigate how
small molecule modifications of the protein surface can
influence that protein’s interaction behaviour. Proteins
labelled with fluorescent dyes are used extensively in protein
interaction studies both in vivo and in vitro. Frequently, such
dyes (e.g. FITC) are bulky, predominantly hydrophobic
molecules. Considering that the labelling procedure is often
non-specific (via amino groups in the case of FITC) we
Figure 1. FITC binding site in
hypothesize that the surface of the labelled protein may be
1flr. Aromatic side chains are
significantly altered, resulting in non-native protein
depicted in sticks. FITC is green.
interactions. Indeed several crystal structures of protein-FITC
complexes (PDB entries: 1n0s, 1t66, 1flr) reveal that FITC is
accommodated in hydrophobic binding pockets (see Figure
1).1 Therefore, we propose to rigorously test the effect of
labelling on protein interaction behaviour.
To make the problem more readily tractable we
propose to study a dimer system. The test molecule is Azurin,
a structurally well-characterised -sheet protein, which can
Figure 2. FITC-Azurin uptake in
invade mammalian cells.2 The crystal structure of Azurin
breast cancer cell line MCF-7.
reveals a dimeric arrangement assembled via the conserved
Nuclei are stained with DAPI.
surface hydrophobic patch.3 The architecture of the dimer
interface makes Azurin an interesting test molecule for these studies. Using selective
labelling via surface exposed cysteine residues,4 bulky hydrophobic molecules (FITC
analogues) will be introduced at different sites in and around the hydrophobic patch.
Biophysical methods including size exclusion chromatography, NMR spectroscopy
and X-ray crystallography will be used to assess the effect on dimer formation.
This project is integral to our investigation of protein interactions. We aim to make
progress in the area of protein-ligand interactions.5 It is envisaged that the successful
completion of the project will lead to at least one publication as well as providing
preliminary data for future grant applications (e.g. RFP). In addition, the results of the
in vitro experiments will be used to inform/interpret our cell uptake studies (Figure 2).
References:
[1] Terzyan et al. J. Mol. Biol. 2004, 339, 1141-1151. [2] Yaamada et al. Proc. Natl.
Acad. Sci. USA 2004, 101, 4770-4775. [3] van Amsterdam et al. Nat. Struct. Biol.
2002, 9, 48-52. [4] Simon et al. Cell 2007, 128, 1003–1012. [5] Crowley et al. Chem.
Bio. Chem. 2008, 9, 1029-1033.