We are starting to fully understand the ‘cofactor story’ as far as the
effect and
importance they have on restriction activity and complex
formation. The
introduction of fluor labelled DNA has made the process
faster and safer to
work with which means data can be collected collated and
reproduced more
accurately.
The molecular modelling predictions supplied by Janusz and those produced
by
myself have started to provide the first structural information on any HsdR
subunit. Although these models
are only predictions and are very often wrong,
they provide a testable
hypothesis of a structure which has started to be
backed by the limited
proteolysis works and the AFM images which have
begun to show a 3D structure
of the protein for the first time in such high
resolution.
The limited proteolysis so far has provided a lot of information about the
domain ‘make-up’ of HsdR and we are currently awaiting data obtained
by
tandem mass spectrometry, on proteolytic fragments isolated from an
SDS-PAGE
gel. This will allow us to
determine the amino acid sequence of each
fragment.
The AFM work will be continued with different cofactors to try to
visualise
structural differences and this work
will be complemented with Scanning
Tunnelling Microscopy (STM).
Janusz is currently working to model the rest of HsdR, especially the
N-terminus
which evidence is building to
suggest is is highly flexible and therefore this will
be a very challenging
task.
I am also working to lay some crystal screens for HsdR in the hope of
gaining
some seed crystals before the end of
my studies.
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