Anders Bergkvist - UspA
Universal Stress Protein A
This project is a rather new NMR project and it is done in cooperation with
Prof. Thomas Nyström. The major NMR work that has been done so far has
been done by a diploma worker under my supervision.
UspA is an E. coli protein (15.9 kDa) which is transcriptionally
induced, when the cell reaches stasis, as a function of various stress factors.
The protein has been cloned and overexpresses all right in rich as well as in
minimal medium (>7 mg/l). We have an (more than two years) old sample grown
on rich medium which show no deterioration. However, we have had some problem
with stability on proteins grown in minimal medium. A possible explanation of
this is a problem with some purification step. Despite this, presumably
transient, problem, we have been able to detect 15N-HSQC spectra
(before the protein deteriorated) which show signal dispersion indicative of a
defined fold. We also know that the protein is a dimer (total weight of 32 kDa)
and that it is overexpressed with one phosphate attached to each monomer, in
rich medium.
In my mind, devising a purification procedure that will leave a stable protein
also in minimal medium shouldn’t be too difficult. After that, a backbone
assignment would require 15N- and 13C-labeling and
possibly 2H-labeling. However, a backbone assignment would then
enable identification of not only secondary structure elements but also of the
phosphorylation site as well as a possible ATP-binding site. Further side-chain
assignments and structure calculations would require special attention to the
dimeric form of the protein, but would still be quite possible.
The structure of UspA would be very interesting since no sequence homologies
have been found to existing structures. The structure would also be very
interesting since the protein have a well-characterized physiological, but not
biochemical, function.
Related publications:
"Cloning, mapping and nucleotide sequencing of a gene encoding a universal
stress protein in Escherichia coli", T Nyström and
F C Neidhardt, Mol. Microbiol. 6 (1992), 3187-3198
"The Universal Stress Protein, UspA, of Escherichia coli is
Phosphorylated in Response to Stasis", Freestone et al., J. Mol. Biol.
274 (1997), 318-324
Last modified: November 17, 1999