Welegedara, Adarshi Priyanga
Description
Site-specific protein labelling presents an important tool for
protein structural biology by
spectroscopic techniques. This thesis focuses on the development
of new spectroscopic
labels and labelling strategies to improve the sensitivity,
accuracy and scope of NMR and
EPR spectroscopy experiments of proteins.
Double electron–electron resonance (DEER) spectroscopy measures
the distance
between two paramagnetic metal centres introduced by
...[Show more] site-specific attachment of
suitable tag molecules. Good DEER tags possess rigid tethers to
position their
paramagnetic centres at a well-defined location relative to the
protein and deliver narrow
DEER distance distributions with high sensitivity, which can
provide accurate
information about protein flexibility. This thesis introduces
new, cyclen-based Gd 3+ tags
and small, Gd 3+ chelating tags designed to deliver narrow DEER
distance distributions.
Chapter 2 describes the development of two cyclen-based
double-arm Gd 3+ tags
designed for binding to the target protein at two and three
points to obtain the narrowest
Gd 3+ –Gd 3+ DEER distance distributions ever recorded with
proteins. It also describes
DEER distance measurements with the iminodiacetic acid tag
attached to cysteine (Cys),
where tags attached to two neighbouring Cys residues combine to
chelate a single Gd 3+
ion. These results have been published in a journal article
(Welegedara et al., Chem. Eur.
J. 2017, 23, 11694−11702).
Chapter 3 discusses two single-armed Gd 3+ tags, a cyclen-based
Gd 3+ tag and a
PyMTA tag that forms a heptadentate Gd 3+ binding motif. Both
tags deliver the shortest
possible tethers to cysteine residues and are shown to produce
narrow DEER distance
distributions in proteins.
For applications in NMR spectroscopy, proteins can be labelled
site-specifically
with NMR probes such as trimethylsilyl (TMS) probes, which
deliver readily detectable
1D 1 H-NMR signals. Introduction of paramagnetic tags and NMR
probes by attachment
to Cys residues requires mutations of native Cys residues to
achieve site-selectivity,
which is not possible with structurally and functionally
important Cys residues. Chapter
4 demonstrates a solution to this limitation by introducing a
selenocysteine (Sec) residue,
which can be selectively reacted with probe molecules at slightly
acidic pH without
iiiaffecting naturally occuring Cys residues. To achieve this, a
Sec residue was introduced
as a photocaged unnatural amino acid (UAA), PSc, to bypass the
otherwise unavoidable
challenges associated with the natural Sec incorporation
mechanism. UV illumination of
PSc yielded Sec with no evidence for the formation of undesired
dehydroalanine
byproducts. Selective tagging of Sec residues with TMS tags was
shown to deliver a
useful tool for studies of ligand binding to proteins. These
results have also been
published in a journal article (Welegedara et al., Bioconjugate
Chem. 2018, 19,
2257−2264).
Site-selective incorporation of isotope-labelled PSc, photolysis
and anaerobic
deselenization opens an indirect route to labelling a single
specific alanine residue in a
protein with stable isotopes. Such samples would have important
applications in
heteronuclear NMR, as they allow the selective detection of the
labelled alanine residue
with maximal spectral resolution. As shown in Chapter 5,
deselenization of
selenoproteins into alanine is possible but requires extremely
anaerobic conditions to
eliminate serine as the main unwanted byproduct.
A range of UAAs has been developed to serve as spectroscopic
probes or facilitate
the introduction of spectroscopic probes via biorthogonal
reactions. The increasing
demand for proteins with different UAAs and the significant cost
of some of these UAAs
has led to increasing popularity of cell-free protein synthesis
(CFPS) systems, which use
amino acids more sparingly than in vivo expression systems.
Mutants of pyrrolysyl-tRNA
synthetase (PylRS)/tRNA CUA pairs have been developed into a
particularly versatile tool
for the incorporation of many structurally different UAAs, but
most produce
disappointedly poor protein yields in vivo. Chapter 6 describes
attempts to develop an in-
house CFPS system with a PylRS/tRNA CUA pair. In addition, the
polyspecific G2
synthetase has been reported to facilitate the incorporation of
sterically demanding UAAs
and Chapter 7 of this thesis describes attempts to develop a CFPS
system with the G2
synthetase.
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