Download Introduction Methods Procedure Conclusion and Future Work

Procedure
Introduction
Protein-protein interactions can vary the structure and function of
cells. A molecule that can bring together two proteins is called a
dimer. Studying the dimerization or proteins can help determine
how protein-protein interactions alters its function as well as the
behavior of cells. This could also be related to cancerous cells. It is
possible that when specific proteins of a cancer cell dimerize, it
could lose its ability to proliferate. We proposed a method in which
a peptide with modified side chains could dimerize proteins.
Synthesis of Dimer
1. Synthesis of the peptide: KKKPPPPPPPPKKK
2. Acylate the C-terminus:
5%
Acetic Anhydride
O
3. Remove the protecting group (Mmt) on lysine residues:
Based on previous work done with polyproline type II helix, we
decided that it would be an ideal linker. It is often called a
“molecular ruler” because it has a pitch of 0.31 nm per residue and
is very rigid, which makes it very strong. If the linker was flexible,
the dimer would be able attach itself to only one protein instead of
two. Depending on the size of the protein, the length of the proline
residues can be adjusted in order for the them to interact.
C
CH2
O
H
R
O
OH
OH
H3N
CH
C
N
OH
O
R
OH
O
O
O
NH2
R
H
O
O
H2O
O
O
NH2
N
Ni2+
N
Histidine
H2O
O
O
O
N
O
N
O
O
O
N
O
H
N
OH
N
H
N
H
NH3+
NH3+
NH3+
O
H
N
N
H
N
O
N
O
N
O
O
N
N
O
N
O
N
O
O
N
O
O
H
N
O
N
H
N
H
O
N
N
O
O-
N
N
O
O
-O
-O
N
N
O
O
O-
O
O
O-
O
O-
-O
O
O
-O
O
O-
O
O-
-O
-O
Conclusion and Future Work
The matrix-assisted laser desorption/ionization time of flight
(MALDI–TOF) was done before and after the modification of the
lysine side chains. The molecular weight of the molecule that was
synthesized matched our predicted values. Therefore we
continued the experiment
We will be using a green fluorescent protein (GFP) containing a
six-histidine tag as the protein of interest. It has a molecular
weight about 27kD, which should work well with our molecule
containing eight prolines. Currently we are in the process of
purifying the modified peptide via high performance liquid
chromatography (HPLC). Once we have our purified molecule and
GFP, we will be able to determine if it is capable of dimerizing the
proteins. We also could also use different proteins or a different
polyproline linker length to test this method of dimerization.
References
OH
O
N
O
O
OH
N
NH
O
O
N
O
O
O
OH
O
N
NH3+
H2N
2
CH
O
N
NH3+
NH3+
Ninhydrin Test
O
IDA can bind metal ions such as nickel. Nickel is capable of
polyvalency, which allows it to bond to six different atoms. It is
often used to purify proteins because it can coordinate with
polyhistidine-tags. Each nickel-IDA can bind two histidines. Since
the protein of interest has a six-histidine tag, we decided that three
IDA are necessary.
H O
H2N
N
5% Triisopropylsilane (TIPS) 1% Trifluoric Acid (TFA) 94% DCM 12:1 tert-­‐butyl bromoacetate 18:1 DIPEA DMF 5. Ninhydrin Test to make sure all of the lysine residues reacted.
(Non-colored indicates all of it reacted)
6. Cleave from the resin bead.
7. Purification via high performance liquid chromatography (HPLC).
8. Allow the protein of interest to interact with the dimer.
O
O
H
N
N
H
4. Modification to make IDA:
Methods
O
O
H2N
8.5% DIPEA
91% DMF
O
O
O
Blue Chromophore
1.  Gaberc-Porekar et al. Perspectives of immobilized-metal affinity. 2001. 335-360.
2.  Grunwald et al. Quantum-Yield-Optimized Fluorophores. 2011. 8090-8092.
3.  Schuler et al. Polyproline and the “spectroscopic ruler”. 2005. 2754-2759.