Structural Model of HsdR

This model of the HsdR subunit was provided by Janusz M Bujnicki – Warsaw,
Poland, allowed us to predict a possible MTase binding site. From the model
we were able to choose amino acids associated with protein protein interaction
between HsdR and MTase. Substitutions made were to change Aspartate and
Glutamate to Alanine residues at key locations. Mutations made by Eva
Sisakova have been designed to disrupt a helix by the introduction of a Proline.
This helix is predicted to stabilise the large flexible region joining the N-terminus
(Left of picture) and the rest of the protein. We believe this will have a significant
effect on the restriction ability of HsdR.

A hybrid HsdR subunit produced from a fusion of EcoR124I and EcoprrI made
by substituting the first 200 amino acids from EcoPrrI:HsdR herein referred
to as HsdR(prrI) contains a cysteine at position 4.
This cysteine has been shown to be freely accessible for attachment through a
malemide link. We have shown the availability through the attachment of Alexa
fluor 633. Limited proteolysis of fluor labelled protein revealed a 40kDa fragment
being released after 130 minutes, through incubation with modified trypsin
(TPCK treated). The N-terminal region predicted in the polish model (see figure
above) is also 40kDa

The availability of the cysteine provided a convenient method for attachment
to a gold surface for visualisation by Atomic Force Microscopy (AFM). AFM
analysis showed the flexibility by stretching the N-terminus through
force wash force drying.
HsdR(PrrI) which was imaged in 8 different directions across this section
of the surface show at least 3 distinct domains. The first of which is
the N-terminal ‘anchor point’ seen as a tail to the right of each of the
protein molecules in the image. The other two domains are seen as two
peaks in the top of the protein with a clear ridge running down the side.

P.S.  Subsequent size measurements of these complexes have shown they
are too big to be HsdR(prrI) - by about 10-fold - therefore they appear
to be conglomerates of protein attached to the surface.

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