Knecht, Steven. Development of new tags for solid-phase peptide synthesis. 2008, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_8400
the preferred binding motifs for Ni2+-NTA. Elongation of such a binding motif led to a
decrease in binding affinity, probably due to increased entropy costs upon binding.
To reduce the entropy costs, short three amino acid tags were designed with
decreased rotational freedom to fix the two histidine residues in the binding
conformation, as it was the case with His-Aib-His. Compared to the His-Gly-His
(KD = 54 μM) the binding affinity could be decreased by a factor of 1.5 with the
aminoisobutyric acid (Aib) at position 2 to a KD of 36 μM.
1,10-Phenanthroline and derivatives thereof show a high potential as a tag binding
to Ni2+-NTA and were analyzed in the Biacore assay. The high binding affinity of
1,10-phenanthroline (KD = 650 nM) is mainly based on a slow dissociation rate
constant (koff) with a half-life time of about 5 min. Different parameters, such as the
charge transfer between the binding nitrogen and nickel, rigidity, and additional
interactions between the binding partners were found to influence the binding affinity.
Among these factors, solubility played the most crucial role. Ligands establishing on
the side directed towards the solvent a well-organized solvation shell, showed
improved binding properties for the Ni2+-surface. Therefore, 5-amino-1,10-
phenanthroline (KD = 407 nm) binds with a 1.6-fold higher affinity to Ni2+-NTA than
1,10-phenanthroline (KD = 650 nM). On the side involved in binding however, a
weaker solvation is desired, because the removal of strongly bound solvent molecules
prior to the binding lowers the gain of enthalpy in the process of the complex
formation. In parallel to the Biacore assay, a computational approach to predict
binding affinities of various ligands to Ni2+-NTA was developed by Dr. Martin
Smiesko, a member of the Institute of Molecular Pharmacy.
To study the phenanthroline-tag and demonstrate its suitability for SPPS, the
following simple strategy was chosen: Phenanthroline was coupled via an acrylate
spacer to the N-terminal of a test peptide. Interestingly, the affinity purification of the
test peptide proved to be superior to the standard HPLC purification method and
afforded a pure product with a yield of 43% compared to 34% for the HPLC
purification. The potential of the new tag could also be demonstrated in a Biacore
assay with the phenanthroline-tagged peptide, where a stable immobilization could be achieved despite the negative influence of the peptide on the binding affinity, which
led to a 20-fold increase in KD.
After Ni2+-NTA purification of a tagged protein, the tag has to be cleaved. For this
purpose, a photolabile linker was introduced between the phenanthroline and the
peptide. Due to the poor solubility, the synthesis of the tag-photolinker construct did
work with only 11% yield. Because only small amounts of the phenanthrolinephotolinker-
construct could be purified, the photolinker approach was abandoned.
Finally, a screen to identify new tags using the Biacore and the computational
model resulted in a promising scaffold, the picolinic acid. The 6-amino-picolinic acid
turned out to be the best representative with a KD of 10.9 μM. The aromatic nitrogen
and one oxygen of the carboxylic acid occupy the two available coordination sites of
Ni2+-NTA. Compared to picolinic acid, the binding affinity was 1.4-fold increased due
to the interaction of the anilinic nitrogen with the carboxylic acid of the NTA chelate.
With this work a new purification strategy using phenanthroline-tags could be
presented. Furthermore, the knowledge about the binding properties of Ni2+-NTA
binding tags was increased. This may add to the development of new tags as presented
for the picolinic acid.
|Committee Members:||Eberle, Alex N.|
|Faculties and Departments:||05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Molekulare Pharmazie (Ernst)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||199|
|Last Modified:||30 Jun 2016 10:41|
|Deposited On:||13 Feb 2009 16:37|
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