1. 87S
α-Synuclein (α-Syn) is an intrinsically disordered (140 aa ~14.5 kDa) monomeric protein that
is abundantly expressed in the neuronal cytoplasm and has been the basis for many
biophysical and biochemical studies due to its accumulation in Lewy bodies, the
histopathological hallmarks of Parkinson’s disease 1. Structurally, α-syn is composed of three
distinct domains including an amphipathic lipid binding N-terminus (residues 1-65), a
central non-Ab component (NAC residues 61-95) responsible for aggregation, and an
unstructured carboxyl-terminal (residues 95-140)2,3. Under certain conditions α-syn displays
the ability to self-associate in a highly dynamic aggregation process where the protein
ultimately polymerises into insoluble filaments, termed amyloid fibrils4. During this
pathway, the protein adopts multiple structural assemblies with the oligomeric form of the
protein contributing largely to neuronal cell death5. As the conformation of a-syn is
dependant on its local environment and interaction partners, the agent that initiates the
aggregation process has been difficult to pinpoint6. Current literature suggests that
phosphorylation may be attributable to the aggregation of α-syn as post-mortem analysis of
PD patients have revealed α-syn to be extensively phosphorylated at Ser129, a residue
located at the C-terminus of the protein7. The exact mechanisms of this process and the
putative kinases involved however, remain unclear. In this study we use NMR spectroscopy
to investigate the potential pathogenic role of a-syn phosphorylation.
Proton and 15N-1H HSQC NMR spectroscopy yields information on enzyme activity, and site specific
changes derived from protein phosphorylation. Although CK2 is documented to phosphorylate
Ser129, this major phosphoaccepter remains unperturbed in our analysis. Minor chemical shifts in
our NMR results that correspond to computationally predicted sites may indicate alternative CK2a
phosphorylation sites. A greater signal intensity of residues within the C-terminus of a-syn is
consistent with the premise that this region remains unstructured even upon aggregation.
•Separate phosphorylated from non phosphorylated a-syn, and analyse by NMR.
•Explore the information provided by carbonyl (CO) 13C-NMR chemical shift data.
•Confirm site specific phosphorylation by Mass Spectrometry.
•Further characterise the effect of phosphorylation on the aggregation of α-syn both in buffer, and a
cell lysate, a more physiologically relevant environment.
•Monitor fibril formation by Thioflavin T (ThT) fluorescence
•Compare the fibril morphology of modified and unmodified a-syn by Atomic Force Microscopy and
Electron Microscopy.
•Compare the site specificity and activity of CK2a to its holoenzyme counterpart.
•Outline a protocol for the expression and purification of the catalytic subunit of the
serine/ threonine protein kinase; Casein Kinase 2.
•In a time dependent manner, monitor the biological activity of the recombinantly
expressed enzyme (CK2a) by its ability to phosphorylate a control peptide using proton
NMR.
•Establish a method for the site specific monitoring of protein phosphorylation by 15N-
1H HSQC NMR.
Introduction Results
Kieran Hand, Dr Jill Madine, Dr Hannah Davies, Dr Marie Phelan
Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK.
References & Acknowledgements
Results
Developing a method to observe the phosphorylation and
aggregation of amyloid proteins using NMR spectroscopy
1 Real-time monitoring of enzyme kinetics by Proton NMR
HSQC NMR: Phosphorylation of a-syn by CK2α
Aims & Objectives
In a reaction comprised of recombinant CK2a and a control peptide (RRRDDDSDDD) acting as
substrate, hydrolysis can be observed by the decrease in ATP signal intensity and the increase of
ADP over the experiment time course.
Figure 2 | a-Syn was incubated in the presence of CK2a and [γ-32P] ATP and the reaction quenched at specific time
points. Experiments were performed in duplicate, and samples that were resolved by SDS-PAGE (A) were either
autoradiographed, (B) Immunoblotted (C), and subject to quantitative analysis by scintillation counting (displayed as
counts per minute) (D).
Figure 1 | Monitoring the catalytic activity of CK2a in a time dependant manner by Proton NMR. 100mM peptide with
200mM ATP in 20mM Tris, 50mM KCl, 10mM MgCl2, pH 7.5 at 30oC over 700 mins.
Dr Mark Wilkinson Dr Helen Wright Dr Pat Eyers
3
15N-1H HSQC NMR was used to investigate the site specific phosphorylation of a-syn in
a time-dependant manner.
ATP
ADP
Start
T0
End
T700
8.527 8.523 8.518 8.514 8.509
1H ppm
T0
T1
T2
T3
T4
T5
T6
T7
With enzyme Without enzyme
T0
T1
T2
T3
T4
T5
T6
T7
0 N-terminus 61 NAC 95 C-terminus 140
Biochemical Analysis: Phosphorylation of a-syn by CK2α2
The viability of a-syn to act as a suitable phosphoaccepter for CK2a was assessed by a
combination of biochemical techniques.
α-synuclein
AutoradiographSDS-PAGE
T0 T5 T15 T30 T60 T120
α-synuclein
kDa
188
38
28
17
14
6
3
62
49
A B
Immunoblot
C T0 T5 T15 T30 T60 T120
C Scintillation CountingD
1H (ppm) 1H (ppm)
15N(ppm)
15N(ppm)
200
60
40
30
20
9
Figure 3 | Superimposition of 15N-1H HSQC spectra of a-syn with (A) or without CK2a (B). Chemical shift differences (C)
and intensities of (D) a-syn resonances from experiment start (T0) and end (T7). A representation of the 3 domains of
a-syn and corresponding residues is also shown.
87S
92T
67G
A B
C
D
Conclusions & Future Work
kDa
1. Toth, G. et al. PloS one 9 (2014).
2. Binolfi, A., Theillet, F. X. & Selenko, P. Biochem Soc Trans 40, 950-954 (2012).
3. Waudby, C. A. et al. PloS one 8 (2013).
4. Knowles, T. P., Vendruscolo, M. & Dobson, C. M. Nature reviews. Molecular cell biology 15, 384-396 (2014).
5. Winner, B. et al. Proceedings of the National Academy of Sciences of the United States of America 108, 4194-4199, (2011).
6. Breydo, L., Wu, J. W. & Uversky, V. N. Biochimica et biophysica acta 1822, 261-285 (2012).
7. Sasakawa, H. et al. Biochemical and biophysical research communications 363, 795-799, (2007).