Supplementary Table 1 – Hydrogen exchange rates for alpha-synuclein at pH 7.4 and a temperature of 15 oC.
Residue
LEU
SER
VAL
ALA
GLU
LYS
THR
ALA
ALA
GLY
THR
VAL
LEU
TYR
VAL
GLY
THR
VAL
HIS
ALA
THR
LYS
THR
LYS
GLN
THR
GLY
GLY
ALA
VAL
THR
GLY
VAL
THR
ALA
VAL
ALA
LYS
THR
GLU
GLY
ALA
8
9
15
17
20
21
22
27
30
31
32
37
38
39
40
41
44
48
50
53
54
58
59
60
62
64
67
68
69
71
72
73
74
75
76
77
78
80
81
83
84
85
20 mM phosphate
kHX(1/s)
±kHX(1/s)
300 mM NaCl
kHX(1/s)
±kHX(1/s)
8.0
15.6
11.6
31.4
15.8
36.1
11.4
69.4
26.1
42.6
8.1
65.3
62.8
18.3
16.5
37.3
19.8
24.7
51.2
20.0
25.2
65.7
21.3
42.5
44.0
24.8
21.7
29.1
14.9
14.4
10.2
72.0
41.3
25.2
20.0
27.4
41.3
23.7
10.9
13.6
12.8
51.4
38.6
33.8
26.2
13.3
14.4
1.5
4.6
2.2
8.6
13.5
10.0
1.2
6.3
1.5
6.4
0.3
6.0
5.5
5.4
3.9
13.0
12.5
5.5
7.3
5.2
7.2
5.7
18.2
8.3
6.4
0.9
1.2
0.7
0.5
1.8
3.9
1.5
1.0
0.5
1.3
1.3
6.2
0.9
1.3
8.8
9.0
5.7
5.0
11.0
2.4
2.0
0.8
1.0
1.3
7.3
14.2
9.7
9.8
0.9
3.1
1.8
0.5
7.9
12.4
1.2
2.7
9.4
8.0
2.4
2.4
GLY
SER
ALA
ALA
THR
GLY
PHE
VAL
LYS
ASP
ASN
GLU
ALA
GLN
GLU
LEU
ASP
ASP
GLU
ALA
MET
SER
GLU
GLU
GLY
ASP
TYR
GLU
ALA
86
87
89
90
92
93
94
95
96
98
103
104
107
109
110
113
119
121
123
124
127
129
130
131
132
135
136
139
140
7.3
9.6
5.1
5.5
7.9
13.0
2.5
0.5
0.5
0.8
2.4
4.0
5.9
5.1
8.1
9.2
2.9
3.0
5.2
3.0
3.6
2.1
4.0
3.3
3.0
3.3
4.1
4.6
5.2
5.3
3.4
2.7
4.4
3.0
0.5
0.3
2.3
3.9
0.3
0.9
0.7
0.8
0.5
0.7
0.8
0.8
0.5
0.6
0.4
0.2
0.6
0.6
0.3
0.8
0.9
0.7
36.9
23.0
44.4
69.9
51.2
33.1
20.1
31.7
28.4
14.3
23.9
8.1
74.6
29.6
64.4
41.3
24.5
9.0
12.7
14.5
23.4
5.1
53.0
8.8
45.2
45.1
21.5
23.0
24.7
4.8
40.7
27.2
Supplementary Fig. S1
Figure S1. NMR assignments for 0.25 mM S in 20 mM sodium phosphate, pH 7.4, and a temperature of 10 oC.
Supplementary Fig. S2
Figure S2. NMR spectra of S without (A-C) or with (D-F) 3.5 mM GM1 micelles. 1H-15N HSQC (A, D), H-C
selective ct-1H-13C HSQC (B, E), and aromatic-selective ct-1H-13C HSQC (C, F). All spectra were recorded at 10 oC.
Positive and negative phases are indicated by black and red contours.
Supplementary Fig. S3
Figure S3. CLEANEX spectroscopy of S. (A) Control Fast-HSQC spectrum. (B) CLEANEX spectrum with a 16 ms
mixing time. The two spectra are plotted so that residues from the acidic C-terminal domain (A107, N113, D119,
A124) have roughly the same number of contours. Plotted this way, the correlations from the first 100 amino acids are
enhanced relative to those from the last 40 amino acids in the CLEANEX spectrum (B) indicating faster hydrogen
exchange for the N-terminal domain. Residue V71, A85 and A89 which are slow exchangers from the N-terminal
domain are exceptions, and can be seen at low contour levels in 1H-15N HSQC spectra of S recorded at pH 7.4 and 35
o
C, 20 mM sodium phosphate (see Fig. 1D of the main text for the V71 correlation).
Supplementary Fig. S4
Figure S4. Representative CLEANEX curves showing the rise in normalized peak intensity due to exchange (20 mM
phosphate sample), as the mixing time that allows magnetization transfer from solvent to sites on the protein is
increased. The initial slopes of these curves are dependent on the rates of hydrogen exchange at particular sites, and are
typically larger for the N-terminal residues (red) than the C-terminal residues (blue).
Supplementary Fig. S5
Figure S5. Conformational exchange contributions to 15N R2 values (R2ex) at (A) 10 oC and (B) 35 oC. The 250 µM
S sample was in 20 mM phosphate buffer at pH 5.9 in order to minimize exchange of the amide protons at the higher
temperature. R2ex values were calculated from the difference in apparent R2s collected with  delays (delays between
sequential 180o 15N pulses in the CPMG train) of 0.625 ms and 20 ms. The CPMG mixing time was 80 ms. The mean
R2ex values were 0.1 Hz at 10 oC and -0.3 Hz at 35 oC indicating the absence of significant exchange contributions at
either temperature. Average errors were 0.6 and 1.3 Hz at 10 oC and 35 oC, respectively. The larger errors at 35 oC are
probably due to residual hydrogen exchange, which attenuates signal intensities but does not interfere with R2
measurements.