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Implications of molecular interactions for protein structure, function and design

Meier, Markus. Implications of molecular interactions for protein structure, function and design. 2005, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7290

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Abstract

Protein structures are kept in a delicate balance of stability by the interactions of the amino
acid residues among themselves, the solvent and other molecules. On one hand they
must be stable enough not to unfold, on the other hand they must be mobile enough to
undergo structural changes if necessary. Only thus they are able to fulfil their various
functions in living organisms, e.g. the catalytic function of an enzyme, protein-ligand recognition
or the rapid reorganisation of the cytoskeleton.
In this work, we have investigated the contributions of such molecular interactions to protein
structure in a functional enzyme, cystathionine [beta]-synthase. We have further analysed
the contribution of ionic interactions to the stability of various designed peptides which
form coiled coils. Finally, we have collected a statistics of electrostatic interactions in
naturally occurring coiled coils to find out which ionic interactions significantly contribute
to a successful formation of coiled coils. The results have important implications for the
design of coiled-coil proteins.
Cystathionine [beta]-synthase is an enzyme of the transsulfuration pathway in eukaryotic cells
which catalyses the condensation of serine and homocysteine to yield cystathionine in a
pyridoxal 5'-phosphate-dependent [beta]-replacement reaction. The human enzyme also contains
heme as a second cofactor which is not required for catalysis. We have solved the
structure of the catalytic domain of human cystathionine [beta]-synthase by X-ray crystallography.
This is the first protein structure containing a heme binding motif where the iron
of the heme is coordinated by a histidine and a cysteine residue. We have also discovered
an oxidoreductase active site motif on the surface which might play a role in enzyme
regulation. There are more than 100 point mutations known in this enzyme which can
cause homocysteinurea disease in humans, characterised by dislocated eye lenses,
skeletal problems, vascular disease and mental retardation. We have mapped the mutations
in the catalytic domain on the structure and were able to find an explanation for the
harmful effect of some mutations by analysing the molecular interactions of the concerned
residues.
Coiled coils are a simple and regular structural motif in proteins consisting of [alpha]-helices
which coil around each other. They can form dimers, trimers, tetramers and pentamers
depending on their amino acid sequence and the environment. The principles and factors
which lead to this specific fold can therefore be studied in detail. The stability of coiled
coils is mainly achieved by the systematic packing of the side chains of the residues at
the interface between the helices, called knobs-into-holes packing. We could show, however,
that a complex network of inter- and intrahelical salt bridges also contributes significantly
to coiled-coil stability by designing short peptides which form dimeric or trimeric
coiled coils. The importance of the ionic interactions could be demonstrated by removing
a single interhelical salt bridge which abolished the formation of the coiled coil. The peptides
were characterised by circular dichroism, analytical ultracentrifugation and X-ray
crystallography.
We have developed the computer program SBSCC to collect a statistics of intrahelical salt
bridges in pure [alpha]-helices and coiled coils from the protein database. We have identified
the salt-bridge configurations that have the highest probability to form the ionic interaction
and which occur most frequently in [alpha]-helices and coiled coils. We have found interesting
differences between [alpha]-helices, parallel and antiparallel 2-stranded coiled-coils with important
implications for the coiled-coil design. We have found a positive correlation between
the probabilities of different salt-bridge configurations to form the ionic interaction
and their frequencies in [alpha]-helices and coiled coils. This indicates that nature relies indeed
on ionic interactions to stabilise [alpha]-helices and coiled coils, an issue which was hitherto
controversially discussed.
Advisors:Burkhard, Peter
Committee Members:Mayans, Olga and Aebi, Ueli
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Structural Biology (Aebi)
Item Type:Thesis
Thesis no:7290
Bibsysno:Link to catalogue
Number of Pages:121
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:41
Deposited On:13 Feb 2009 15:15

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