Figure S1 . Analytical HPLC profiles (

= 214 nm) together with ESI MS data (inset) corresponding to each major product of crude synthetic peptides: (a) N-terminus segment and (b) C-terminus segment, of
L-plectasin.
Figure S2.
Analytical HPLC profiles (

= 214 nm) together with ESI MS data (inset) corresponding to each major product of crude synthetic peptides: (a) N-terminus segment and (b) C-terminus segment, of
D-plectasin.

Figure S3.
Purified L-plectasin synthetic peptide segments: analytical HPLC profiles (

= 214 nm) together with ESI MS data (inset) corresponding to the purified (a) N-terminus segment (obs. 2249.0 Da, calc. 2249.3 Da (av isotopes)) and (b) C-terminus segment (obs. 2363.6 Da, calc. 2363.8 Da (av isotopes).
The minor earlier eluting peak in spectrum (a) corresponds to oxidized product containing Met(O).
Figure S4.
Purified D-plectasin synthetic peptide segments: analytical HPLC profiles (

= 214 nm) together with ESI MS data (inset) corresponding to the purified (a) N-terminus segment (obs. 2249.0 Da, calc. 2249.3 Da (av isotopes)) and (b) C-terminus segment (obs. 2363.6 Da, calc. 2363.8 Da (av isotopes)).

Figure S5.
Chemical synthesis of L-plectasin: analytical HPLC profiles (

= 214 nm) together with ESI
MS data (inset) corresponding to each major product for the ligation and folding. The chromatographic separations were performed using a linear gradient (5

65%) of buffer B in buffer A over 15 min (buffer
A = 0.1% trifluoroacetic acid (TFA) in water; buffer B = 0.08% TFA in acetonitrile) on a C-4 (Vydac),
2.1

150 mm column at 40 o
C. (a) Native chemical ligation of (Gly
1 
His
18
)
 
COSCH
2
CH
2
Ala-COOH
( 1 ) and (Cys
19
-Tyr
40 
COOH ( 2 ) at t = 3 min, 3 indicates the formation of a transthioesterified intermediate with 4-mercaptophenylacetic acid, 4 is the ligation product (Gly 1 
Tyr 40 ). (b) Native chemical ligation at 3 h showing the complete disappearance of 2 and formation of the ligation product 4 .
(c) Folding of the crude full-length linear L-peptide after solid phase extraction of the deformylated product. Folding was essentially complete within 3 h; the folded protein had an earlier elution time and a mass decrease of 34 Da compared to the formylated linear peptide; this corresponds to loss of formyl (28
Da) and the formation of three disulfides (loss of 6 Da). Asterisks (in a-b) indicate 4mercaptophenylacetic acid.

Figure S6. Chemical synthesis of D-plectasin: analytical HPLC profiles (

= 214 nm) together with ESI
MS data (inset) corresponding to each major product for the ligation and folding. The chromatographic separations were performed using a linear gradient (5

65%) of buffer B in buffer A over 15 min (buffer
A = 0.1% trifluoroacetic acid (TFA) in water; buffer B = 0.08% TFA in acetonitrile) at 40 o
C. (a) Native chemical ligation of (Gly
1 
His
18
)
 
COSCH
2
CH
2
Ala-COOH ( 1 ) and (Cys
19
-Tyr
40 
COOH ( 2 ) after 15 min, 3 indicates the formation of a transthioesterified intermediate with 4-mercaptophenylacetic acid
(MPAA), 4 is the ligation product (Gly
1 
Tyr
40
). (b) Native chemical ligation products after 3 h. (c)
Folding of the crude full-length linear D-peptide after solid phase extraction of the deformylated product.
Folding was essentially complete within 2 h; the folded protein had an earlier elution time and a mass decrease of 34 Da compared to the formylated linear peptide; this corresponds to loss of formyl (28 Da) and the formation of three disulfides (loss of 6 Da). Asterisks (in a-b) indicate MPAA. In-house packed
C-4, 2.1

100 mm column (in a ) with flow rate 0.4 mL/min and C-4 (Vydac), 2.1

150 mm column (in b and c ) with flow rate 0.5 mL/min was used for the chromatographic separation.

60
0
-20
40
20
L-plectasin
D-plectasin
-40
-60
208 218 228
Wavelength (nm)
238 248
Figure S7. Circular dichroism spectra of L-plectasin and D-plectasin. CD was recorded at room temperature using 131.7 µM of L-plectasin and 136.3 µM of D-plectasin solution in water. CD cuvette path length = 0.1 cm. Data shown 250-208 nm. Total number of scans = 10.

Figure S8.
Data for minimum inhibitory concentration (MIC) assay of plectasin enantiomers using
Staphylococcus aureus newman (MSSA).

Figure S9.
Data for minimum inhibitory concentration assay (MIC) of plectasin enantiomers using
Staphylococcus aureus USA300 (MRSA) and Bacillus cereus.

Table S1.
The X-ray data collection and refinement statistics for the crystal structures of plectasin.
Data collection
Space group
Cell dimensions
 a , b , c (Å)
      
(

)
Mol/asymmetric unit
Mol/unit cell
DL-plectasin*
P

1
23.6, 23.8, 24.5
109.7, 95.0, 97.7
L-plectasin
P6
1
54.0, 54.0, 22.0
90.0, 90.0, 120.0
Resolution (Å)
R merge
**
I /

I
Completeness (%)
1
2 (one D- and one Lmolecule)
50.00 - 1.00 (1.04 –
1.00)
0.068 (0.212)
12.0 (4.8)
83.2 (32.0)
1
6
50.00 - 1.35
(1.40 – 1.35)
0.076 (0.428)
23.3 (5.2)
99.1 (97.0)
Redundancy
Refinement
Resolution (Å)
No. reflections
R work/
R free
3.5 (2.2) 8.7 (7.5)
22.8 - 1.0 (1.03-1.00) 23.4 - 1.35
21051
0.202 / 0.224
Completeness (working + test, %) 83.2 (30.4)
(1.39-1.35)
7731
0.157 / 0.182
99.2 (96.1)
No. atoms
Protein
Water
Average B-factor (Å 2 )
R.m.s deviations
Bond lengths (Å)
Bond angles (º)
309
24
11.8
0.02
1.75
295
48
26.5
0.02
1.74
Number of crystals = 1 for each structure.
*The structure of the racemic plectasin was not refined with bulk solvent corrections due to the low solvent content of the crystal.
** R merge
= SUM ( ABS(I - <I>)) / SUM (I).
***Highest resolution shell is shown in parenthesis.

Table S2 I/Sigma in resolution shells for the L-plectasin structure collected to 1.2 Å resolution
Lower
Limit
50.00
2.59
2.05
1.79
1.63
1.51
1.42
1.35
1.29*
1.24*
All hkl
Shell
Upper
Limit
2.59
2.05
1.79
1.63
1.51
1.42
1.35
1.29
1.24
1.20
I/Sigma in resolution shells
% of of reflections with I / Sigma less than:
0
0.0
0.2
0.5
1.6
1.6
3.2
5.0
8.4
10.2
11.3
4.2
1
0.2
0.7
1.3
2.9
3.6
7.4
11.7
18.3
22.7
31.2
9.9
2
0.3
0.9
2.4
5.6
6.1
14.3
21.2
32.1
39.9
49.6
17.0
3
0.4
1.3
3.5
7.7
9.2
22.3
30.5
43.8
55.2*
64.0*
23.3
5
0.7
2.1
5.8
11.9
16.0
31.9
46.8
63.5
73.2
80.5
32.8
10
2.2
4.6
12.4
22.3
33.0
54.9
72.8
87.8
91.1
95.5
47.1
20
21.6
13.5
30.8
52.9
69.2
87.4
95.0
98.8
96.4
97.2
65.8
>20
77.8
86.4
68.7
46.5
29.9
11.1
3.1
0.9
0.2
0.0
33.0
Total
99.4
99.8
99.5
99.4
99.1
98.5
98.1
99.7
96.6
97.2
98.7

Table S3. Hydrogen bond lengths r (Å) at the interface between L-plectasin and D-plectasin enantiomers in space group P

1 shown in Figure 3A-3B
[a]
.
Protein Residue config. number
Residue detail r (Å) Residue detail
Residue number
Protein config.
L
Donor
38 LYS-NZ 2.58 PRO-O
Accepter
7 D
L
L
38
29
LYS-NZ
TYR-OH
2.86
2.50
GLU-OE1 10
GLU-OE2 10
D
D
Protein Residue config. number
Residue detail
Mediated by water molecule r (Å) Water r (Å) Residue detail
Residue Protein number config.
L 40 TYR-OXT 2.90 w2 2.73 TRP-NE1
2.86 CYS-O
8
30
D
D
L 32 LYS-N 2.70 w1 2.82
3.05
ASP-OD1
ASP-OD2
12
12
D
D
[a] Two enantiomers forming the interface are related by a center of symmetry. Each hydrogen bond interaction occurs twice at the interface. Only one half of the L-D pairs are listed. The second half may be derived by inverting the protein configuration.

Table S4. Hydrogen bond lengths r (Å) at the interface between two L-plectasin molecules in space group P6
1
shown in Figure 3C-3D.
Protein config.
Residue number
Residue detail r (Å) Residue detail
Residue Protein number config.
L
Donor
32 LYS-NZ
2.83
2.79
3.41
2.94 CYS-O
Acceptor
GLU-OE2 10
ASP-OD1 12
ASP-OD2 12
30
L
L
L
L
Protein Residue config. number
Residue detail
Mediated by water molecule r (Å) Water r (Å) Residue detail
Residue Protein number config.
L
L
38
32
LYS-NZ
LYS-O
3.19
2.89 w1 w2
2.68
3.22
ASP-OD2
TYR-OH
12
29
L
L
L 10 GLU-OE1 2.64
GLY-O w3
2.85 w4 [ a]
3.08 TYR-OH 40 L
L 33 w5 [ a]
2.68 TYR-OH 29 L
[a]
Two hydrogen-bonded (2.76 Å) waters w4 and w5 mediate contact between Gly33-O and Tyr29-OH.