S1

Supporting Information

Visualizing Proton Antenna in High Resolution Green Fluorescent Protein Structure

Ai Shinobu, Gottfried J. Palm*, Abraham J. Schierbeek and Noam Agmon*

Table S1. X-ray data collection and refinement statistics for GFP sg11

Data collection

X-ray source Bruker Microstar

Montel multilayer optic

Wavelength (Å) 1.5418

Resolution limits (Å) (highest shell) 68 -0.90 (1.00-0.90)

Space group P212121

Unit cell parameters a b c (Å ) 52.15 59.42 68.11

Unique reflections 155426 (41798)

Redundancy 19.7(9.2)

Completeness (%) 91.4 (90.8)

I/σ(I) 20.4 (3.0)

Rmerge 0.080 (0.487)

Wilson B factor (Å2) 8.4

Solvent content (%) 39

Refinement

Resolution limits (Å) 6 - 0.90

Total reflections 133320

No. reflections in test set 4231

Rcryst 0.1456

Rfree 0.1740

No. amino acid residues in a.u. 229

No. water/other solvent molecules in a.u. 326

Rmsd bond lengths (Å) 0.034

Rmsd bond angles (deg.) 3.4

PDB entry code 2WUR

S2

Table S2. List of PDB codes for GFP and mutants structures. Green shading indicates a continuous ASW without a break at Ser72, whereas blue shading indicates structures belonging to type B (see text).

PDB

code

Resolution

[Å] Mutations*

Monomers

per

asymmetric

unit **

1 1BFP 2.1 Y66H/Y145F 1

2 1C4F 2.25 S65T 1

3 1CV7 2.5 K26R/F64L/S65T/Y66W/N146I/M153T/V163A/

N164H 1

4 1EMA 1.9 S65T 1

5 1EMB 2.13 wt 1

6 1EMC 2.3 F64L/I167T 4

7 1EME 2.5 F64L/I167T 1

8 1EMF 2.4 F64L/Y66H/V163A 1

9 1EMG 2.0 S65T 1

10 1EML 2.3 F64L/I167T 1

11 1EMM 2.3 F64L 1

12 1F09 2.14 S65G/V68L/S72A/H148Q/T203Y 1

13 1F0B 2.1 S65G/V68L/S72A/H148Q/T203Y 1

14 1GFL 1.9 wt 2

15 1HCJ 1.8 wt 4

16 1HUY 2.2 S65G/V68L/Q69M/S72A/T203Y 1

17 1JBY 1.8 S65T/H148G/T203C 1

18 1JBZ 1.5 S65T/H148G/T203C 1

19 1KYR 1.5 F64L/Y66H/F99S/Y145F/H148G/M153T/V163A 1

20 1KYS 1.44 F64L/S65T/Y66H/F99S/Y145F/H148G/M153T/V163A 1

21 1MYW 2.2 F46L/F64L/S65G/V68L/S72A/M153T/V163A/

S175G/T203Y 1

22 1OXD 1.15 F64L/S65T/Y66W/N146I/M153T/V163A 1

23 1OXE 1.15 F64L/S65T/Y66W/N146I/M153T/V163A 1

24 1Q4A 1.45 S65T 1

25 1Q4B 1.48 S65T 1

26 1Q4C 1.55 S65T/T203C 1

S3

27 1Q4D 1.58 S65T/T203C 1

28 1Q4E 1.38 S65T/Y145C 1

29 1Q73 1.6 S65T/Y145C/T203C 1

30 1QXT 2.0 F64L/S65T/R96A/F99S/M153T/V163A 1

31 1QY3 2.0 F64L/S65T/R96A/F99S/M153T/V163A 1

32 1QYF 1.5 F64L/S65T /R96A/F99S/M153T/V163A 1

33 1QYO 1.8 F64L/S65G/Y66G/F99S/M153T/V163A 1

34 1QYQ 1.8 F64L/S65G/Y66G/F99S/M153T/V163A 1

35 1RM9 2.9 F64L/S65T/Y66W/N146I/M153T/V163A 1

36 1RMM 1.9 F64L/S65T 1

37 1RMP 3.0 F64L/S65T 1

38 1RRX 2.1 F64L/S65T 1

39 1S6Z 1.5 F64L/S65T/Y66L 1

40 1W7S 1.85 wt 4

41 1W7T 1.85 wt 4

42 1W7U 1.85 wt 4

43 1YFP 2.5 S65G/V68L/S72A/T203Y 2

44 1YHG 2.5 F64L/S65G/Y66S/V68G/F99S/M153T/V163A 2

45 1YHH 1.5 F64L/S65A/Y66S/G67A/F99S/M153T/V163A 1

46 1YHI 1.9 S65A/Y66S/R96A/F99S/M153T/V163A 1

47 1Z1P 2.0 F64L/S65T/Y66L 1

48 1Z1Q 1.5 F64L/S65T/Y66L 1

49 2AWJ 1.6 F64L/S65T/R96M/F99S/M153T/V163A 1

50 2AWL 1.85 F64L/S65T/R96K/F99S/M153T/V163A 1

51 2AWM 1.7 F64L/S65T/R96A/F99S/M153T/V163A/Q183R 1

52 2B3P 1.4 S30R/Y39N/F64L/S65T/F99S/N105T/Y145F/

M153T/V163A/I171V/A206V 1

53 2DUE 1.24 S65T 1

54 2DUF 1.5 S65T/H148D 1

55 2DUG 1.4 S65T/H148N 1

56 2DUH 1.2 S65T/H148N 1

57 2DUI 1.36 Q80R/H148D 1

58 2EMD 2.0 F64L/Y66H 1

59 2EMN 2.3 F64L/Y66H 1

60 2EMO 2.6 F64L/Y66H/V163A 1

61 2G16 2.0 S65A/Y66S/F99S/M153T/V163A 1

62 2G2S 1.2 F64L/S65G/Y66S/F99S/M153T/V163A 1

S4

63 2G3D 1.35 F64L/S65G/Y66A/F99S/M153T/V163A 1

64 2G5Z 1.8 S65G/Y66S/F99S/M153T/V163A 1

65 2H6V 1.47 F64L/S65T/T203Y 1

67 2H9W 1.82 F64L/S65T/T203Y 1

68 2HGD 1.6 S65A/Y66F/F99S/M153T/V163A 1

69 2HGY 2.05 S65A/Y66F/F99S/M153T/V163A/E222A 1

70 2HJO 1.25 F64L/S65T/V224H 1

71 2HQZ 1.2 L42H/F64L/S65T 1

72 2O24 1.45 F64L/S65T/T203Y 1

73 2O29 1.8 F64L/S65T/T203Y 1

74 2O2B 1.94 F64L/S65T/T203Y 1

75 2OKW 1.9 F64L/S65T/S205C 6

76 2OKY 2.4 F64L/S65T/S205C 2

77 2Q57 2.0 F64L/S65T/Y66W/S72A/Y145A/N146I/

H148D/M153T/V163A 1

78 2QLE 1.59 Q80R/S205V 4

79 2YFP 2.6 S65G/V68L/S72A/H148G/T203Y 1

80 3CBE 1.31 C48S/F64L/F99S/S147C/H148S/M153T/

V163A/I167T/Q204C 1

81 3CD9 1.5 C48S/F64L/F99S/S147C/H148S/V163A/

I167T/Q204C 1

82 2WUR 0.9 F64L/I167T 1

* Mutations Q80R, K238N and the Ala insertion after the N-terminal Met are cloning artifacts and

not explicitly mentioned. They do not affect the spectral properties of GFP.

** In PDB entries with more than one monomer per asymmetric unit, each monomer was used

independently for structural comparisons, with the exception of 1YFP- chain A, 1YHG- chain A,

2OKW- chain C, and 2QLE- chain B. This gives a total of 104 monomers.

S5

Table S3. Statistical properties of hydrogen-bonded clusters for the 104 PDB structures of GFP

mutants listed in Tbl. S2.

Property y Average STD

Correlation

coefficient

with

resolution

Correlation

coefficient

with water

fraction

Linear fit parameters for water

fraction (fw):

y = A + B fw

A B

Number of

clusters 22.5 8.6 -0.624 0.720 9.09 ± 1.41 149.96 ± 14.32

Maximal cluster

size 63.4 48.4 -0.664 0.762 -16.29 ± 7.37 892.07 ± 74.91

Average cluster

size 14.0 5.2 -0.647 0.741 5.73 ± 0.82 92.44 ± 8.29

Total number of

protein atoms

that participate in

clusters

193.4 88.0 -0.829 0.965 9.82 ± 5.41 2053.99 ± 55.01

Total number of

water oxygens

that participate in

clusters

135.0 83.6 -0.796 0.984 -42.71 ± 3.53 1988.29 ± 35.91

Total number of

atoms that

participate in

clusters *

328.4 169.9 -0.820 0.984 -32.89 ± 7.23 4042.27 ± 73.49

* protein + waters

S6

sg11- based reference atom list:

The partition of the ASW into sub-clusters was done here differently than in the main text (Tbl. S4 below). Atoms were assigned to the cluster in which they are present in more than 50% of the GFP structures. This is reflected mainly in the cut near Ser72 which divides sub-clusters 2 and 3. In addition, 10 atoms from the exit end were deleted because they participate in very few structures. Table S4: Partition of the sg11-based reference atom list into sub-clusters. From this 71-atom list, 10 atoms were deleted from cluster 3 (these are depicted in purple in Fig. S1).

sub-cluster 1 sub-cluster 2 sub-cluster 3

O GLU 5 Oε1 GLU 5 Oγ SER 65

O GLU 6 Oε2 GLU 5 Oη TYR 66

O LEU 7 O GLN 69 N VAL 68

O PHE 8 O CYS 70 O VAL 68

O THR 9 O SER 72 Nε2 GLN 69

Oγ1 THR 9 Oγ SER 72 O PHE 71

N GLY 10 N TYR 74 O TYR 143

O GLY 10 Nζ LYS 79 O TYR 145

N VAL 11 O LYS 79 O ASN 146

O VAL 11 O ASP 82 N SER 147

O GLU 34 Oδ1 ASP 82 Oγ SER 147

Oε1 GLU 34 Oδ2 ASP 82 Nδ1 HIS 148

Oε2 GLU 34 Nζ LYS 85 O HIS 148

Oδ1 ASP 36 Oγ1 THR 203

Oδ2 ASP 36 O THR 203

N ALA 37 O GLN 204

O ALA 37 N SER 205

N THR 38 O SER 205

Oγ1 THR 38 Oγ SER 205

Oη TYR 39 N LEU 207

Oγ1 THR 43 O LEU 207

O ASP 117 Oε1 GLU 222

Oδ1 ASP 117 Oε2 GLU 222

Oδ2 ASP 117

Oγ1 THR 118

S7

Figure S1. Schematic depiction of the connectivity in the sg11 active-site cluster (PDB file 2WUR). Atoms are colored according to the partition into sub-clusters in Tbl. S4: red- sub-cluster 1, blue- sub-cluster 2, green- sub-cluster 3, purple- sub-cluster 4 that was not considered in the sub-cluster analysis due to its scarcity within the 103 PDB structures. Note that O-T203 and N-S147 are in green because in structures other than sg11 (e.g., 1EMB), they usually belong to sub-cluster 3.

S8

The division into sub-clusters enables us now to correlate separately the number of atoms in each

sub-cluster with the water fraction of the various GFP structures. As can be seen, the correlation

exhibited in the main text for the total ASW holds for every sub-cluster separately.

Figure S2. Correlation graphs for the number of atoms from sg11-based list (Tbl. S4) appearing in a

specific structure vs. its water fraction. Graphs are shown for sub-clusters 1, 2 and 3 and for the

combined list of 71 atoms. (The latter is the same as Fig. 6B in the main text). Linear fit data presented

in Tbl. S5.

S9

Table S5. Statistical properties of the number of protein atoms participating in the hydrogen-bonded

sub-clusters from the list in Tbl. S4 for the 104 PDB structures of GFP mutants.

Number of

protein

atoms (y)

present in

GFP

mutants in:

Average STD

Correlation

coefficient

with

resolution

Correlation

coefficient

with water

fraction

Linear fit parameters for water

fraction (fw): y = A + Bfw

(See Fig. S2)

A B

Sub-cluster

11 15.08 7.73 -0.782 0.902 0.01 ± 0.78 168.56 ± 7.98

Sub-cluster

22 9.51 3.21 -0.813 0.772 4.15 ± 0.48 59.94 ± 4.89

Sub-cluster

33 14.23 4.51 -0.592 0.602 8.35 ± 0.85 65.77 ± 8.62

Total sg11-

based

reference

list 4

41.07 14.86 -0.826 0.909 11.87 ± 1.46 326.58 ± 14.87

1 Out of 25 atoms 2 Out of 13 atoms 3 Out of 23 atoms 4 Out of 71 atoms. Sub-clusters 1, 2, 3 combined + 10 additional deleted atoms (colored purple in Fig. S1)

S10

A similar analysis is possible for water oxygen atoms (instead of protein atoms) in each sub-

cluster. These correlations are summarized in Tbl. S6 below. Note that sub-cluster 1, which is on a

hydrophilic surface patch, has the highest water fraction of all sub-clusters.

Table S6. Statistical properties of the number of water oxygen atoms participating in the hydrogen-

bonded sub-clusters from the list in Tbl. S4 for the 104 PDB structures of GFP mutants. Because the

clusters of each PDB structure are not identical to the 2WUR clusters, the counting of the waters was

done as follows: for each PDB structure, in each sub-cluster, if an atom from the reference list appeared

in one of that PDB structures clusters, we counted all the water oxygen atoms which are H-bonded to

that atom, and summed over all the atoms in the sub-cluster.

Fraction of

water

oxygens (y)

in:

Average STD

Correlation

coefficient

with

resolution

Correlation

coefficient

with water

fraction

Linear fit parameters for

water fraction (fw):

y = A + Bfw

A B

Sub-cluster 1 0.306 0.168 -0.806 0.931 -0.032 ± 0.014 3.780 ± 0.146

Sub-cluster 2 0.260 0.102 -0.803 0.808 0.081 ± 0.015 1.996 ± 0.144

Sub-cluster 3 0.231 0.101 -0.619 0.750 0.066 ± 0.016 1.837 ± 0.160

Total sg11-

based reference

list

0.270 0.122 -0.806 0.937 0.024 ± 0.010 2.758 ± 0.102

S11

Figure S3. ASW fragments of a decarboxylated wt-GFP: (A) At 100K (PDB file 1W7T, A subunit);

(B) After annealing at 170 K (PDB file 1W7U). Hydrogen bonds are coded so that black bonds - exist in

both structures, blue bonds - exist only in 1W7U, red bonds - exist only in 1W7T. It is clearly seen how

annealing leads to formation of the Thr203-His148 exit pathway and to the restructuring of 3 water

molecules near Ser72, that now connect it with Ser65.