Travelling wave ion mobility mass spectrometry-based conformational studies of prion protein – effects of metal cation binding and buffer
gas
1
1
1
1
1
1
2
Susan E. Slade , Konstantinos Thalassinos , Gillian R. Hilton , Narinder Sanghera , Teresa Pinheiro , Claudia A Blindauer , Michael T. Bowers , James H. Scrivens
1
1
2
Synapse
plasma
membrane
Determine the effect of different gases used in the travelling wave mobility cell on the observed
cross-sections.
pH 5.5
+
H
INTRODUCTION
Fourier transform infra red studies have shown characteristics of the two forms of PrP can be
C
related to their differences in structure. PrP is predominantly á-helical in content whereas,
Sc
PrP has a higher proportion of â-sheet [3] (Figure 1). This change in the core structure either
as a single monomer or via a dimeric structure, is characteristic of a potential disease state.
-1
2
10000
5000
betaPrP
beta-PrP90–231
90–231
0
-5000
-10000
-15000
200
220
240
260
Wavelength
Wavelength // nm
nm
Alpha
2300
The experiments were repeated at acidic pH 5.5 (data not shown), no differences were
observed between the data sets using all the different mobility gases, demonstrating the
reproducibility of the data.
Beta
2100
reduction to Cu (I)?
transport by chaperones?
5500
5500
1500
2+
Figure 3: Working hypothesis for PrP function in Cu transport via endocytosis. At extracellular
2+
pH, each octarepeat cooperatively binds a single Cu . Within the endosome (at pH 5.5),
protonation of the amides competes with Cu2+ coordination thereby lowering the affinity for the
metal ion. Adapted from Burns. C. S. et al. [7]
MATERIALS AND METHODS
Recombinant SHa PrP (23-231) and (90-231) were expressed in Escherichia coli strain BL21*
Rosetta. The prion protein was purified using the conditions described by Mehlhorn et al., [8,
9] (respectively) and folded in a predominately á-helical form. The secondary structure was
determined by far-UV circular dichroism (CD).
2+
The relative cross-sections of SHa PrP (23-231) both in the presence and absence of Cu
have been calculated using all of the mobility gases described earlier. Figure 9 shows the
cross-sections obtained under nitrogen (b) and carbon dioxide (a). Under all of the conditions
analysed, no difference in cross-section was observed (within experimental error) upon
copper binding, independent of the mobility gas used.
1900
1700
endosome
Prion diseases are a class of fatal, infectious neurodegenerative diseases that affect the
central nervous system in both humans and animals [1]. These diseases include bovine
transmissible spongiform encephalopathy in cows and Creutzfeldt-Jakob disease in humans
[2]. The infectious agent (PrPSc) is thought to be a conformational isoform of the host-encoded
prion protein. The conversion of the non-infectious cellular isoform (PrPC) to the â-sheet rich
PrPSc and its subsequent aggregation is considered to be the cause of neuronal death.
alpha
alpha-PrP
PrP 90–231
90 –231
6
8
10
12
Charge state
14
5000
16
Figure 4a: Difference in cross section between á and â SHa PrP (90-231) at pH 5.5.
Figure 4b (insert): Far-UV CD spectrum of Sha PrP (90-231) preparations
The relative cross-sections for the a-rich truncated and full length SHa PrP were determined
under physiological and acidic pH, Figure 5. Under these conditions the lower charge state
species (most compact) from both protein species, exhibited very similar or near identical
cross-section conformers despite a difference in molecular weight of over 6 kDa. At higher
charge states, as the protein unfolds, the cross-sections differ significantly, as expected.
Estimated cross-section (Å2)
Calculate relative cross-sections for metal-free and metal-bound prion protein.
2500
15000
4500
Nitrogen
4000
Helium
3500
Carbon
dioxide
Krypton
5500
a
5000
Estimated cross-section (Å2)
endocytosis
2700
b
20000
SHa PrP (23-231) samples containing various concentrations of zinc and manganese (2+)
have been analysed. To date we have not observed a zinc:PrP protein species although we
have observed PrP with one and two Mn ions bound (data not shown).
Estimated cross-section (Å2)
GPI anchor
/ deg
[ cm
q]
Estimated cross-section (Å2)
PrP folded
C terminus
dmol
2
Use of travelling wave-based ion mobility mass spectrometry to characterise the binding of
biologically relevant metal cations to Syrian hamster recombinant prion protein (SHa PrP).
a
2900
O
2+
Samples containing SHa PrP (23-231) at pH 7.0 were analysed using the mobility buffer gases
described previously, Figure 7. Minor variation in relative cross-section was observed in
comparison to those obtained using nitrogen with ±2% and ±3% difference in cross-section
between krypton and carbon dioxide respectively. The relative cross-section appears to be
slightly smaller when helium was the mobility gas. The overall difference in cross-section
between the data collected under nitrogen and helium is ±5%, with the exception of charge
state +10.
25000
1-
Cu
3100
PrP (23-59)
[q] / deg cm dmol
OVERVIEW
pH 7.4
ct
ar
e
Pr pe
P a
(6 t d
0- o
91 m
) ain
University of Warwick, Coventry, UK; University of California, Santa Barbara, CA
4500
4000
3500
3000
2500
2000
1500
b
5000
4500
0 Cu
4000
1 Cu
3500
2 Cu
3 Cu
3000
4 Cu
2500
2000
1500
8
12
16
20
24
28
8
12
Charge state
3000
16
20
24
28
Charge state
Figure 9: Comparison of relative cross-section upon Cu (II) ion binding to á SHa PrP (23-231) at
pH 7 using carbon dioxide (a) and nitrogen (b) as the mobility buffer gas.
2500
(b)
Figure 1. Schematic representation of the NMR-predicted conformational differences between (a)
PrPC 47% á-helix/3% â-structure and (b) PrPSc 17-30% á-helix/45% â-structure [3].
The physiological function of PrPC is still not fully understood. Prusiner et al. observed PrP
deficient mice develop normally but show resistance to prion infection [4]. Proposed prion
functions include cell adhesion, antioxidant, signal transduction and a metal transporter [5].
Studies by Brown et al. suggest that the prion protein is able to bind copper in vitro [6], with
further investigations revealing the favoured binding site to be the octapeptide repeat unit
found on the N-terminus (Figure 2).
Q Glutamine
W
Tryptophan
G G
N
P
I
Isoleucine
R
L
Leucine
Lysine
V
Valine
R Arginine
G
Glycine
H Histindine
A
Alanine
Cysteine
S
Serine
T
Threonine
P
Proline
K
C
M Methionine
F
Phenylalanine
50
S
G
Y
P
Q
P
G G
G
G
S
T
W
N
R
Cu2+
W G
P
Q
G
G G
Y
P
G
Q
T
Proteinase K
cleavage site
G
P
W G
Q
Cu2+
G
P
G
H
H G
W
G
W G
Cu2+
Q
G
P
G
Cu2+
Q
H G
450
100
10.8
Nitrogen
9
300
30
5.5
Carbon
dioxide
8
300
24
3.9
Krypton
8
300
5
1.5
Xenon
10
400
2
1.0
Table 1: Summary of IMMS conditions used for various gases
Copper (II) sulfate (Sigma Aldrich, Poole, UK) stock solutions were prepared in 10 mM
ammonium formate (Acros Organics, New Jersey, USA) pH 5.5. The stock solutions were
diluted and mixed with a SHa PrP (23-231) to achieve the final stoichiometric ratios of 1:1, 1:3,
and 1:5 (SHa PrP (23-231): Cu(II)). The sample introduced into the mass spectrometer
contained 10% methanol with the pH brought to 7.0 with 0.1 M ammonia.
Q
G
G
G
G
T
Cu2+
Octarepeats
Relative cross-sections were obtained by calibrating the Synapt against published cross
sections of horse heart myoglobin [10] as described previously [11-12]
H
N
Q
W
G
A
A
V
A
V
A
G
A
G
G
A
M
H
K
M
N
T
K
P
K
S
P
L
125
K
2500
2000
6
11
16
Charge State
21
16
4000
3500
24
CONCLUSIONS
28
90-231
23-231
3000
2500
2000
6
26
20
Charge state
1500
1500
12
11
16
Charge State
21
26
Figure 5: Comparison of relative cross sections for SHa PrP 23-231 and 90-231 at pH 5.5 (a) and
at pH 7 (b).
The IMS mobility gas was changed from the standard configuration of nitrogen to the inert
gases helium, krypton, xenon and carbon dioxide to assess the effect of gas on the observed
cross-section. After equilibration of the mobility cell in the appropriate gas, a calibration was
created using bovine cytochrome C which was used to calculate the relative cross-sections of
various charge states from equine myoglobin. Figure 6 shows a comparison of the observed
relative cross-sections plotted against the published cross-sections [10]. The observed crosssections are in good agreement with ±2% deviation of the estimated cross-section from the
published data.
Figure 7: Comparison of various mobility gases to observe the overall differences in cross-section
of á SHa PrP (23-231) at pH 7.
The binding of metal cations to the prion protein is still a contentious issue. It is now generally
accepted that PrP (23-231) does bind copper (2+) with between 2 and 6 copper ions per
monomeric unit being reported. The binding of metal cations has been implicated in the
2+
conversion to the misfolded disease state [5]. We monitored the binding of Cu to SHa PrP
(23-231) at physiological pH using various concentrations of PrP to copper, from 1:1 to 1:5
molar equivalents, Figure 8. At a 1:1 ratio, PrP species were observed that have one and two
2+
copper ions bound. At 1:3 and 1:5 (PrP:Cu ) concentrations, the prion protein without copper
was almost absent from the spectrum with the prion protein containing 3 copper ions bound
the most abundant observed species. Data acquisition on the highest concentration copper
sample was difficult due to aggregation of the protein:copper complex resulting in blockages
of the nanoflow capillaries, consistent with the observations of Requena et al. [13]. We were
unable to collect data on samples containing greater than 5 copper molar ratio equivalents as
immediate aggregation occurred.
PrP + 4 Cu
PrP + 2 Cu
N
100
0
Q
C- terminus
Figure 2: Schematic representation of the Syrian hamster prion protein residues 23-125 with
copper binding sites indicated.
Understanding metal interaction with prion protein may help to elucidate its physiological role
(Figure 3) and the mechanism associated with conversion from PrPC to PrPSc. SHa PrP has
been analysed by ion mobility mass spectrometry (IMMS). IMMS is a rapid, gas phase
separation technique that yields information regarding molecular shape. This technique has
successfully and reproducibly differentiated between conformational variants of the same
C
protein under acidic and physiological pH. Studies into copper binding with recombinant PrP
species have been carried out and conformational measurements made.
RESULTS
Far-UV CD was used to characterise each form of SHa PrP prior to mass spectrometry
analysis. The á SHa PrP (90-231) spectrum shows the typical features of a protein containing
a high á-helical structure with well defined minima at 208 nm and 222 nm. In contrast, the
spectrum for refolded SHa PrP (90-231) produced minima at 216 nm indicating an increase in
â-sheet structure (Figure 4b).
A significant difference in the relative cross-sections was observed using nitrogen mobility
gas, between predominately á and â-rich SHa PrP (90-231) species at pH 5.5 (Figure 4a) that
was not observed under pH 7 conditions (results not shown).
3500
1:3 PrP:Cu
PrP + 2 Cu
0
Nitrogen
PrP + 1 Cu
Helium(Optimised)
Xenon
2000
100
PrP
PrP + 2 Cu
1:1 PrP:Cu
0
1500
PrP
100
1000
1000
1500
2000
2500
3000
No Cu added
3500
0
2
Published cross-sections (Å )
Figure 6: Comparison of relative and published cross sections for equine myoglobin charge states
+6 to +16 calibrated against cytochrome C bovine using different mobility gases.
•
•
•
•
[1] Prusiner, S. B. (1997). Science 278(5336): 245-51.
[2] Collinge, J. and M. S. Palmer (1992). Curr Opin Genet Dev 2(3): 448-54.
[3] Pan K.M., et al. Proc Natl Acad Sci U S A, (1993). 90(23): 10962-10966.
[4] Prusiner, S. B., D. Groth, et al. (1993). Proc Natl Acad Sci U S A 90(22): 10608-12.
[5] Choi, C. J., A. Kanthasamy, et al. (2006). Neurotoxicology 27(5): 777-87.
[6] Brown, D. R., K. Qin, et al. (1997). Nature 390(6661): 684-7.
[7] Burns. C. S., et al.,(2002), Biochemistry, 41, 3991-4001
[8] Mehlhorn I., et al. (1996) Biochemistry, 35(17): 5528-37
[9] Sanghera N., et al.(2002) J Mol Biol, 315(5): 1241-1256.
[10] http://www.indiana.edu/~clemmer/Research/cross%20section%20database/Proteins/protein_cs.htm
[11] Ruotolo, B. T., Giles, K., et al., (2005). Science 310: 1658-61.
[12] Scrivens, J.H. et al., (2007). Proc. 55th ASMS Conf. Mass Spectrometry
[13] Requena, J. R., et al (2001) Proc Natl Acad Sci U S A, 98(13) 7170-7175.
CO2
2500
•
REFERENCES
PrP + 3 Cu
100
Krypton
•
23050
23100
23150
23200
23250
23300
Travelling wave-based ion mobility mass spectrometry is a powerful method for the study
of conformational changes in recombinant prion proteins.
Relative cross-sections may be easily obtained by calibration against previously published
drift cell-based experimental cross sections.
Small, but significant, differences were observed between predominately á and â-rich SHa
PrP (90-231) species at pH 5.5 but not at pH 7.0.
The observed relative cross-sections for the truncated and full length PrP are very similar
at low charge states, indicating that the N-terminal sequence may not be as unstructured
as predicted.
As the protein unfolds at higher charge states, the relative cross-sections of the truncated
and full length PrP differ, as would be expected due to differences in their respective
relative molecular masses of 6860 Da.
The binding of up to four copper ions to á SHa PrP (23-231) has been observed .
Binding of copper or manganese does not significantly change the relative cross-section of
the á SHa PrP (23-231) protein, casting doubt on proposed mechanism of the N-terminus
becoming more structured on copper binding.
The observed relative cross-section of the á SHa PrP (23-231) protein in the presence of
copper is independent of the mobility gas used.
Minimal interactions between the mobility buffer gas and the protein species were
observed.
1:5 PrP:Cu
4000
3000
•
•
PrP + 3 Cu
100
Helium
•
20 uM alpha PrP (23-231) 10mM Ammonium formate 10%MeOH pH7
G
G
P
G
H G
G
W
8
3000
8
%
Asparagine
Tyrosine
R
P
K
Helium
3500
4500
%
E Glutamate
N
Y
K
Indicated gas
pressure (mbar)
4000
2000
b
%
D Aspartate
23
Indicated gas
Wave
Velocity (m/s) flow (mL/min)
4500
Estimated cross-sections (Å2)
pe
e
r
a
Hex
K
ats
N - terminus
Gas
Wave
Height (V)
a
%
The instrument was operated in mobility mode and the travelling wave IMS cell conditions
optimised for each of the gases nitrogen, helium, krypton and carbon dioxide, see Table 1.
5000
5000
Estimated cross-section (Å2)
All experiments were performed in a hybrid quadrupole/ion mobility/orthogonal acceleration
time-of-flight mass spectrometer (Synapt HDMS, Waters, Manchester, UK). The instrument
was operated with a nanoflow ESI source at 90 °C. The sample solutions were introduced into
the source region by direct infusion operated in positive ion mode. Mass spectra were
acquired at an acquisition rate of two spectra/sec with an interscan delay of 100 ms. Data
TM
acquisition and processing were carried out using MassLynx (v4.1, SCN 566) software
(Waters Corp., Milford, MA, USA).
Estimated cross-section (Å2)
(a)
23350
23400
23450
mass
mass
Figure 8: Deconvoluted mass spectra obtained at different Cu (II) molar ratios bound to á SHa PrP
(23-231) at pH 7 using nitrogen as a mobility gas
ACKNOWLEDGEMENTS
The authors would like to thank Defra and FSA for funding provided. We would also
like to thank Prof. John Ellis for extensive discussions regarding the project.