Apply
here: https://jobs.le.ac.uk/vacancies/9767/research-associate.html

(Please
send initial enquiries to either Hanna Kwon, hanna.kwon@leicester.ac.uk or Andrew Hudson, andrew.hudson@leicester.ac.uk). Deadline is 6^th
September for applications. 

We have an amazing group of people @LISCB and MAXIV Check us out! https://le.ac.uk/research/institutes/structural-chemical-biology

Project
Description

It is
becoming clear that the cell biology of haem is wider than its role as a
prosthetic group in housekeeping proteins[1]. Haem might not always be
inextricably linked to a host, or pivotal to a protein’s functional activity.
One example is its ability to modulate the behaviour of transcription factors,
such as those that generate the internal-timekeeping system of the
mammalian-molecular clock. This type of haem-protein interaction must be
transient and reversible, in contrast to the tight binding of a prosthetic
group. Whilst certain sequences of amino acids have been implicated as
haem-recognition motifs, there is still uncertainty about how haem binds to
transcription factors. The reversible nature of the interaction suggests that
the binding sites must be altogether different to the binding pockets in
haemoproteins. The binding of haem to the transcription factor can be expected
to induce a significant conformational change which might prevent its
association to DNA (or cause an existing DNA-protein complex to dissociate). In
addition, as a consequence of the toxicity of free molecules of haem, we must
assume that transcription factors acquire haem via ligand-substitution
reactions[2] from a chaperone; a protein that is
suspected to moonlight as a haem chaperone is GAPDH[3]. This project will investigate
transient haem-protein interactions by utilizing structural biology to reveal
the ligand-binding site, and different biophysical approaches to reveal the
conformational dynamics of the transition between the apo and holo
protein, along with mechanistic detail of haem-substitution reactions from an
exemplar chaperone, GAPDH to an acceptor protein. 

The
project will be supervised by a team of researchers from the Leicester Institute
for Structural and Chemical Biology (LISCB, Leicester, UK) and MAX IV
Laboratory (Lund, Sweden): Hanna Kwon (Molecular & Cell Biology & LISCB)
will supervise
protein expression and protein crystallography for structural determination of holo-proteins.
Hanna has been working with dioxygenases[4] (relatively weak haem-binding
proteins) for a number of years, and there is evidence in the literature that some
of these acquire haem from GADPH[3]; this would be the appropriate
model system to start looking at the haem substitution reaction. Andrew
Hudson (Chemistry & LISCB) will supervise experiments to study the
dynamic interactions of protein, or protein-DNA complexes, with haem, and haem
transfer reactions from GAPDH to transcription factors using fluorescence
lifetime and fluorescence correlation spectroscopy[2]. Peter Moody (Molecular
& Cell Biology) is experienced in exploiting large-scale European (and
World) infrastructures for structural and mechanism determination, notably with
haem enzymes[5] and GAPDH[6], and will advise on structural
determination of proteins using X Ray, neutron and free electron laser sources.
Kajsa Sigfridsson Clauss (Balder beamline, MAX IV Laboratory) is an expert in X ray absorption
and emission spectroscopy with focus on metalloproteins. These techniques have
been used before to probe the local structure and electronic state of the iron
centre in haem[7-9] and yield more precise and reliable
information than can be obtained by UV-visible absorption spectroscopy (which
is currently used to speculate on the coordination of the iron centre in haem[10]). Kajsa will be a formal member of
the supervisory team, and host the postdoctoral researcher on their secondment
to MAX IV, which will together with beamtime applications secure access to the
Balder beamline for the research work.

1.            Shimizu, T., et al., Heme: emergent roles of heme in signal
transduction, functional regulation and as catalytic centres. Chem Soc Rev,
2019. 48(24): p. 5624-5657.

2.            Leung, G.C., et al., Unravelling the mechanisms controlling heme
supply and demand. Proc Natl Acad Sci U S A, 2021. 118(22).

3.            Biswas, P., Y. Dai, and D.J. Stuehr,
Indoleamine dioxygenase and tryptophan
dioxygenase activities are regulated through GAPDH- and Hsp90-dependent control
of their heme levels. Free Radic Biol Med, 2022. 180: p. 179-190.

4.            Basran, J., et al., Binding of l-kynurenine to X. campestris
tryptophan 2,3-dioxygenase. J Inorg Biochem, 2021. 225: p. 111604.

5.            Kwon, H., et al., XFEL Crystal Structures of Peroxidase
Compound II. Angew Chem Int Ed Engl, 2021. 60(26): p. 14578-14585.

6.            Tourigny, D.S., et al., Expression, purification, crystallization
and preliminary X-ray analysis of wild-type and of an active-site mutant of
glyceraldehyde-3-phosphate dehydrogenase from Campylobacter jejuni. Acta
Crystallogr Sect F Struct Biol Cryst Commun, 2011. 67(Pt 1): p. 72-5.

7.            Yan, J.J., et al., Resonant inelastic X-ray scattering
determination of the electronic structure of oxyhemoglobin and its model
complex. Proc Natl Acad Sci U S A, 2019. 116(8): p. 2854-2859.

8.            Baker, M.L., et al., K- and L-edge X-ray Absorption Spectroscopy
(XAS) and Resonant Inelastic X-ray Scattering (RIXS) Determination of Differential
Orbital Covalency (DOC) of Transition Metal Sites. Coord Chem Rev, 2017. 345: p. 182-208.

9.            Wilson, S.A., et al., X-ray absorption spectroscopic investigation
of the electronic structure differences in solution and crystalline
oxyhemoglobin. Proc Natl Acad Sci U S A, 2013. 110(41): p. 16333-8.

10.          Kuhl, T., et al., Analysis of Fe(III) heme binding to cysteine-containing heme-regulatory
motifs in proteins. ACS Chem Biol, 2013. 8(8): p. 1785-93.

11.          Freeman, S.L., et
al., Heme binding to human CLOCK affects
interactions with the E-box. Proc Natl Acad Sci U S A, 2019. 116(40): p. 19911-19916.