Protein-Ligand Interactions by NMR and EPR Spectroscopy
Date
2016
Authors
Abdelkader, Elwy
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Abstract
Pulse electron paramagnetic resonance (EPR) distance measurements
using double electron-electron resonance (DEER) experiments have
been established as a powerful tool in structural biology. DEER
experiments have the ability to measure the distance between two
paramagnetic centres in biological macromolecules in the range of
about 2 to 8 nm. The paramagnetic centres are usually introduced
into proteins by site-directed spin labelling (SDSL) of cysteine
residues.
This thesis is based on the use of new lanthanide binding tags
(LBTs) for paramagnetic nuclear magnetic resonance (NMR)
spectroscopy (reported in papers 2 and 5), DEER distance
measurements (reported in papers 1 and 3) and time-resolved
luminescence resonance energy transfer (LRET) experiments
(reported in paper 4). In particular, use of two complementary
techniques, DEER experiments and paramagnetic NMR spectroscopy,
was investigated for the study of conformational changes of
proteins as a result of protein-ligand interactions. Two proteins
were studied, the E. coli aspartate/glutamate binding protein
(DEBP) and human calmodulin (CaM). Both proteins have different
ligand binding characteristics: DEBP binds to small organic
molecules, while CaM binds to specific peptide sequences.
DEBP is a periplasmic binding protein responsible for the
transport of aspartic acid and glutamic acid across the cell
membrane and widely used in the design of biosensors of
glutamate. The protein is composed of two domains, which bind one
amino acid molecule at the domain interface. As DEBP contains a
disulfide bond, an alternative cysteine-independent approach for
site-specific protein tagging was used, which involved the use of
genetically encoded unnatural amino acids that were
site-specifically incorporated into proteins using orthogonal
amber-suppressor tRNA/aminoacyl-tRNA synthetase systems.
p-azido-L-phenylalanine (AzF) residues were incorporated into
DEBP at different positions and paramagnetic lanthanide tags were
attached to AzF via Cu(I)-catalyzed click chemistry (papers 1 and
2). Multiple Gd3+-Gd3+ distances measured by DEER experiments
were used to define the metal positions, subsequently allowing
deltachi-tensor determinations from sparse sets of pseudocontact
shifts (PCSs). Both the DEER data and PCSs were in agreement with
the closed conformation observed in the crystal structure of the
homologue from S. flexneri. On the other hand, the PCSs indicated
that the transition to the substrate-free protein involves a
movement of the two domains as rigid entities relative to each
other.
CaM is a two-domain protein that acts as an intermediate
messenger protein and intracellular calcium sensor, which
responds to changes in Ca2+ concentrations by large
conformational changes that enable binding to a range of
different proteins involved in signalling pathways. The
conformational changes of CaM upon binding of the myristoylated
alanine-rich C-kinase substrate (MARCKS) peptide were studied
using DEER experiments and paramagnetic NMR. MARCKS was chosen
due to its unique binding mode compared to other CaM-target
peptide complexes. The DEER results indicated that the binding of
MARCKS peptide to CaM does not lock CaM in a single conformation.
Deviations between the crystal and solution structure of the
complex were also evident in the measured PCS data, highlighting
the conformational flexibility of CaM that allows CaM to bind to
diverse target proteins.
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Double electron–electron resonance, NMR spectroscopy, lanthanide-binding tags, pseudocontact shifts, click chemistry
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