Table 1. Summary of our top designs.
The WT control shows that both the UPLC and FRET experiments underreport the amount of BB homodimer formed, compared to the theoretical 25% if both the A and B chains expressed equally well. The percentages reported for the UPLC experiments represent the fraction of the dimeric species; free monomer species are not included in the tally, as they could be more easily purified away and/or adjusted by changing the transfection ratio of the two chains. Standard errors are reported in parentheses for experiments performed more than once. In the 1:1 UPLC experiments, our best designs, 7.8.60 and 20.8.34, perform roughly as well as the positive controls (KH, DD-KK, and ZW1). For each design, only chain A mutations are given, but looking at each design and its inverse gives mutations for both chains.
| DSC | UPLC 1:1 | UPLC 4:1 | FRET 1:1 | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Design | A Chain Muts | N | Tm | N | AAL | AA | AB | BB | N | AAL | AA | AB | BB | N | AA | AB | BB |
| WT | - | 1 | 82.9 | 12 | 1.6 (0.5) | 31.3 (1.7) | 51.3 (0.6) | 15.9 (1.5) | 3 | 2.3 (0.7) | 65.0 (2.3) | 27.4 (0.9) | 2.9 (0.3) | 13 | 24.2 (1.4) | 61.2 (1.5) | 14.6 (2.5) |
| DD-KK | K409D K392D | 1 | 68.8 | 2 | 1.4 (0.5) | 0.5 (0.2) | 91.7 (0.4) | 6.3 (0.2) | 6 | 3.6 (0.6) | 19.9 (3.3) | 37.5 (4.6) | 2.6 (0.4) | 4 | 2.3 (0.5) | 96.2 (1.0) | 1.6 (0.6) |
| DD-KK−1 | D399K E356K | 2 | 1.8 (0.1) | 1.2 (0.4) | 93.6 (0.2) | 3.3 (0.1) | 2 | 9.6 (1.3) | 11.3 (1.2) | 29.4 (2.3) | 3.5 (0.6) | ||||||
| KH | T366W | 1 | 69.9 | 4 | 3.2 (0.8) | 0.8 (0.2) | 92.4 (1.0) | 3.6 (0.2) | 3 | 14.2 (3.5) | 15.7 (4.0) | 35.0 (7.5) | 2.2 (1.0) | 4 | 2.0 (0.2) | 96.2 (0.7) | 1.7 (0.6) |
| KH−1 | T366S L358V Y407A | 2 | 2.6 (0.2) | 0.8 (0.1) | 94.1 (0.2) | 2.4 (0.1) | 1 | 6.4 | 4.2 | 33.1 | 6.5 | ||||||
| ZW1 | T350V T366L K392L T394W | 1 | 81.2 | 3 | 1.3 (0.4) | 0.6 (0.3) | 94.3 (0.7) | 3.8 (0.4) | 3 | 2.1 (0.8) | 5.1 (2.5) | 18.3 (0.7) | 3.5 (1.3) | 2 | 3.5 (0.2) | 94.8 (0.1) | 1.6 (0.3) |
| ZW1−1 | T350V L351Y F405A Y407V | 2 | 2.4 (0.7) | 1.4 (0.4) | 87.6 (5.2) | 8.6 (5.4) | 2 | 8.6 (2.0) | 9.0 (1.6) | 25.6 (1.3) | 19.4* (18.0) | ||||||
| 7.8 | D399M Y407A | 1 | 71 | 6 | 1.5 (0.2) | 0.5 (0.1) | 89.9 (1.9) | 8.2 (1.9) | 1 | 4.9 (2.6) | 9.4 (3.7) | 42.3 (8.1) | 1.9 (0.5) | 4 | 5.8 (2.1) | 89.0 (2.0) | 5.5 (1.9) |
| 7.8−1 | T366V K409V | 6 | 3.8 (0.4) | 4.1 (1.2) | 88.6 (1.0) | 3.5 (0.4) | 2 | 24.1 (14.6) | 28.6 (7.6) | 26.9 (6.1) | 2.4 (0.6) | ||||||
| 7.8.60 | K360D D399M Y407A | 1 | 70.4 | 1 | 3.9 | 2.3 | 92.9 | 0.9 | 1 | 9.8 | 16.8 | 46.2 | 0.8 | ||||
| 7.8.60−1 | E345R Q347R T366V K409V | 3 | 2.0 (0.3) | 0.4 (0.0) | 93.3 (1.3) | 4.3 (1.4) | 2 | 5.4 (3.6) | 26.4 (6.0) | 32.5 (4.9) | 3.2 (0.4) | ||||||
| 20.8 | Y349S K370Y T366V K409V | 4 | 70.0 | 6 | 4.0 (1.1) | 2.2 (1.0) | 84.9 (1.4) | 6.0 (2.0) | 1 | 1 | 8.4 | 11.7 | 12 | 5 | 3 (2.3) | 93.3 (3.2) | 3.7 (1) |
| 20.8−1 | E357D S364Q Y407A | 2 | 0.2 (0.1) | 0.3 (0.1) | 92.5 (0.1) | 7.0 (0.0) | 1 | 1 | 3.4 | 4.7 | 38.6* | ||||||
| 20.8.34 | Y349S K370Y T366M K409V | 1 | 68.9 | 4 | 3.0 (0.4) | 1.3 (0.2) | 83.8 (3.5) | 3.5 (0.8) | 3 | 9.5 | 12.0 | 23.6 | 30.3* | 4 | 2.3 (0.7) | 93.9 (1.5) | 3.7 (1.6) |
| 20.8.34−1 | E356G E357D S364Q Y407A | 1 | 1.3 | 1.2 | 94.2 | 3.3 | 6 | 3.8 | 4.3 | 31.8 | 3.8 | ||||||
| 20.8.37 | Y349S K370Y T366M K409V | 1 | 68.9 | 2 | 2.3 (0.3) | 0.2 (0.2) | 83.5 (1.7) | 4.8 (1.3) | 1 | 7.9 | 11.9 | 34.7 | 1.5 | ||||
| 20.8.37−1 | E357D S364R Y407A | 1 | 2.3 | 0.7 | 85.5 | 4.6 | 4 | 10.4 | 1.8 | 29.9 | 3.6 | ||||||
In some 4:1 UPLC assays, a prominent peak appeared where the second of the two BB homodimer peaks ought to have been; this likely represents a proteolyzed A monomer species whose peak has shifted significantly later.