Table 1.
Details of HumRadA mutants and summary of binding data
| Mutant | Mutations (all constructs carry also deletion of the L2 loop) |
Tm (°C) | BRC4 affinity (nM)/FP |
RadA-OP affinity (μM)/ITC |
RAD51-OP affinity (μM)/ITC |
|---|---|---|---|---|---|
| HumRadA1 | I169M,Y201A, V202Y, K221M | 94.3 | n.d. | 1.8 ± 0.5 | 24 ± 5 |
| HumRadA2 | I169M, Y201A, V202Y, E219S, D220A, K221M | 82.1 | weak | 3.4 ± 0.3 | n.d. |
| HumRadA3 | I169M, Y201A, V202Y, E219S, D220A, K221M, I222M, V223M, V232Y | n.d. | n.d. | n.d. | n.d. |
| HumRadA4 | I169M, Y201A, V202Y, E219S, D220A, K221M, I222M, deletion of 227–231,V232Y, K233A |
n.d. | n.d. | n.d. | n.d. |
| HumRadA5 | I169M, Y201A, V202Y, L213Q, V215L, Q216Y, E219S, D220A, K221M, D267M, L274E, Y275F |
80.8 | weak | n.d. | n.d. |
| HumRadA14 |
V168A, I169M, W170Y, Y201A, V202Y, L213Q, V215L, Q216Y, E219S, D220A, K221M, I222M, K223V, L225S, V232Y, H264F, D267M, L274E, Y275F |
78.4 | 670 ± 12 | n.d. | n.d. |
| HumRadA16 | V168A, I169M, W170Y, Y201A, V202Y, L213Q, V215L, Q216Y, E219S, D220A, K221M, I222M, K223V, L225S, V232Y, K263R, H264F, A266R, D267M, L274E, Y275F |
77.2 | 294 ± 6 | n.d. | n.d. |
| HumRadA18 | V168A, I169M, W170Y, K198D, H199N, I200V, Y201A, V202Y, L213Q, V215L, Q216Y, E219S, D220A, K221M, I222M, K223V, L225S, V232Y, K263R, H264F, D267M, L274E, Y275F |
76.1 | 10.7 ± 0.35 | n.d. | n.d. |
| HumRadA20 | V168A, I169M, W170Y, K198D, H199N, I200V, Y201A, V202Y, L213Q, V215L, Q216Y, E219S, D220A, K221M, I222M, K223V, L225S, V232Y, K263R, H264F, A266R, D267M, L274E, Y275F |
75.5 | 3.90 ± 0.15 | n.d. | n.d. |
| HumRadA22 | V168A, I169M, W170Y, I182L, K198D, H199N, I200V, Y201A, V202Y, L213Q, V215L, Q216Y, E219S, D220A, K221M, I222M, K223V, L225S, V232Y, K263R, H264F, A266R, D267M, L274E, Y275F | 74.0 | 6.20 ± 0.30 | n.d. | n.d. |
| HumRadA33 | S167K, V168A, I169M, W170Y, N175G, I182L, R183L, D192S, P193G D194S, E195D, K198D, H199N, I200V, Y201A, V202Y, L213Q, V215L, Q216Y, E219S, D220A, K221M, I222M, K223V, L225S, V232Y, K263R, H264F, A266R, D267M, L274E, Y275F | n.d. | n.d. | 14 ± 2 | 0.8 ± 1 |
| HumRadA22F | V168A, I169M, W170Y, I182L, K198D, H199N, I200V, Y201A, V202Y, K221M |
n.d. | n.d. | n.d. | n.d. |
| HumRadA26F | S167K, V168A, I169M, W170Y, N175G, I182L, R183L, K198D, H199N, I200V, Y201A, V202Y, E219S, K221M, I222M, |
n.d. | n.d. | n.d. | n.d. |
| HumRadA28F | S167K, V168A, I169M, W170Y, N175G, I182L, K198D, H199N, I200V, Y201A, V202Y, L213Q, V215L, Q216Y, E219S, K221M, I222M, K223V, L225S, V232Y, K263R, H264F, A266R, D267M, L274E, Y275F |
n.d. | n.d. | n.d. | n.d. |
| HumRadA33F | S167K, V168A, I169M, W170Y, N175G, I182L, R183L, D192S, P193G D194S, E195D, K198D, H199N, I200V, Y201A, V202Y, E219S, K221M, I222M, K223V, V232Y |
n.d. | n.d. | n.d. | n.d. |
A list of all the humanised RadA mutants described in the article, with the mutations they carry (numbering as per PfRadA sequence) and the details of thermal stability (measured by DSF) and affinities towards BRC4 and RadA and RAD51 oligomerisation (OP) peptides, measured by ITC. It is worth noting the decreasing thermal stability as humanisation progresses and the increasing affinity towards BRC4 and RAD51-OP peptides. Mutations introduced for the first time are highlighted in bold. n.d. – not determined.