| | 1stA | | 2ndA | 3rdA | 4thA | OthersA |
|---|
| | | Function % (5-Helix) | Display % (Anti-FLAG) | Function/DisplayC | | Function % (5-Helix) | Display % (Anti-FLAG) | Function/DisplayC | | Function % (5-Helix) | Display % (Anti-FLAG) | Function/DisplayC | | Function % (5-Helix) | Display % (Anti-FLAG) | Function/DisplayC | Function % (5-Helix) | Display % (Anti-FLAG) | Function/DisplayC |
|---|
G28
|
G
B
|
38
|
34
| 1.1 |
N
|
25
|
21
| 1.2 |
D
|
19
|
20
| 1.0 |
S
|
19
|
25
| 0.8 |
0
|
0
| - |
Y30* |
H
|
22
|
9
| 2.4 |
S
|
13
|
4
| 3.2 |
R
|
13
|
11
| 1.2 |
G
|
11
|
9
| 1.2 |
41
|
67
| 0.6 |
H31
|
R
|
23
|
5
| 4.6 |
S
|
19
|
4
| 4.8 |
G
|
15
|
5
| 3.0 |
N
|
14
|
16
| 0.9 |
29
|
70
| 0.4 |
W32
|
S
|
26
|
14
| 1.9 |
N
|
19
|
7
| 2.7 |
R
|
16
|
8
| 2.0 |
H
|
14
|
11
| 1.3 |
25
|
60
| 0.4 |
Y49
|
S
|
60
|
45
| 1.3 |
Y
|
40
|
55
| 0.7 | | - | - | - | | - | - | - | - | - | - |
K50* |
R
|
61
|
10
| 6.1 |
N
|
15
|
18
| 0.8 |
H
|
13
|
23
| 0.6 |
S
|
7
|
17
| 0.4 |
4
|
32
| 0.1 |
S52
|
S
B
|
77
|
49
| 1.6 |
Y
|
23
|
51
| 0.4 | | - | - | - | | - | - | - | - | - | - |
S53
|
R
|
54
|
19
| 2.8 |
H
|
22
|
17
| 1.3 |
S
B
|
11
|
22
| 0.5 |
N
|
8
|
16
| 0.5 |
5
|
26
| 0.2 |
Y91
|
R
|
36
|
13
| 2.8 |
N
|
20
|
17
| 1.2 |
S
|
18
|
15
| 1.2 |
Y
B
|
14
|
24
| 0.6 |
12
|
31
| 0.4 |
S92
|
S
B
|
34
|
30
| 1.1 |
A
|
27
|
20
| 1.4 |
Y
|
27
|
30
| 0.9 |
D
|
12
|
20
| 0.6 |
0
|
0
| - |
N93
|
N
B
|
31
|
28
| 1.1 |
D
|
18
|
10
| 1.8 |
A
|
17
|
13
| 1.3 |
T
|
17
|
28
| 0.6 |
17
|
21
| 0.8 |
Y94* |
Y
B
|
71
|
20
| 3.6 |
T
|
13
|
21
| 0.6 |
S
|
6
|
17
| 0.4 |
N
|
5
|
11
| 0.4 |
5
|
31
| 0.2 |
L96
|
H
|
35
|
14
| 2.5 |
F
|
19
|
15
| 1.3 |
Y
|
19
|
19
| 1.0 |
L
B
|
15
|
25
| 0.6 |
12
|
27
| 0.4 |
- A For each position, the percentage of the population of analyzed clones is shown for the 1st, 2nd, 3rd, and 4th most frequently observed residue from the 5-Helix selection. The WT D5 residue is indicated prior to the position number in the first column and the “hotspot” positions identified by Da Silva et al. are marked by asterisk. If two of the amino acids exhibit the same frequency, the one with the higher Function/Display ratioC will rank in a higher order. In cases where additional substitutions were permitted and observed, these were binned into a 5th category labeled ‘other’. At positions 49 and 52, only two residues (Tyr and Ser) were permitted. The data shown here were compiled from 178 sequences from the 5-Helix selection and 169 sequences from the display selection.
- B Amino acids shown in the four most frequently observed at each position among library members that are identical to WT D5 are shown in gray italic font. For positions that the WT residue was not encoded in the library due to codon degeneracy, amino acids that have the closest physiochemical properties to the WT that is among the four most frequent residues are in black italic fonts.
- C Ratio of percent frequency observed from functional (5-Helix) selection and percent frequency observed from display (anti-FLAG) selection.
