Table 1.
Summary of results
| Mutants | Complementation
|
Mutant
proteins
|
Mutant in cdc3 ts strain
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Rep81
|
Rep3
|
Urea (M) | PLP
|
Nucleotide
exchange
|
Polymerization
|
Expression level 36°C 4 h (μg/10 mg protein) | Average length/ width 36°C 12 h (nm) | Increased cell density 36°C 12 h (fold) | ||||
| Null
|
Ts
|
Ts
|
||||||||||
| 25°C | 32°C | 36°C | 36°C | Affinity fraction of wild typea | Affinity fraction of wild typea | Acceleration fraction of wild typea | fraction of wild type affinitya | |||||
| Controls | ||||||||||||
| Wild type | Yes | Yes | Yes | Yes | 4.5 | (55 μM) | (0.21 μM)b | (2.3-fold) | 1.0 | 0.67 | 12.4/3.9 | 9.0 |
| C89S | Yes | Yes | Yes | NT | 3.2 | 1.1 | 1.0b | 1.0 | NT | 0.53 | 13.0/3.9 | 8.9 |
| E42K | Yes | No | Yes | Yes | 2.2 | NT | NT | NT | NT | N/D | 18.6/4.7 | 6.7 |
| PLP binding mutants | ||||||||||||
| L121A | NT | Yes | Yes | NT | NT | 0.11 | NT | NT | NT | 0.36 | 14.7/4.5 | 9.2 |
| W30Y | NT | Yes | Yes | NT | 4.5 | 0.32 | NT | NT | NT | 0.28 | 14.7/4.6 | 8.1 |
| Y5A | Yes | Yes | Yesc | NT | 4.0 | 0.26 | 0.7b | 0.77 | NT | 0.53 | 21.3/5.4 | 6.0 |
| Y5D | No | No | No | No | 3.3 | 0.011 | 0.68b | 1.3 | 0.5 | 0.60 | 25.5/5.9 | 4.5 |
| Y120A | NT | No | Yes | NT | 4.1 | 0.037 | 0.15b | 1.1 | NT | 0.44 | 20.6/5.5 | 6.0 |
| Y120D | No | No | Yes | NT | 3.9 | 0.031 | 1.4b | 0.92 | NT | 0.66 | 22.9/5.6 | 5.4 |
| L121R | No | No | No | NT | 1.6 | NT | NT | NT | NT | NT | NT | NT |
| Y126D | No | No | Yesc | NT | 4.1 | 0.031 | 0.31b | 1.2 | NT | 0.41 | 17.3/4.7 | 4.7 |
| Actin binding mutants | ||||||||||||
| K67A | No | No | Yesc | NT | 4.4 | 0.91 | <0.01 | 0 | <0.025 | NT | 15.1/4.7 | 7.5 |
| K67E | No | No | Yesc | Yesc | 4.2 | 1.0 | <0.01 | 0 | <0.025 | NT | 22.3/5.9 | 5.1 |
| K67L | No | No | Yesc | Yes | P/F | NT | NT | NT | NT | NT | NT | NT |
| K67W | No | No | No | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| I69W | No | No | Yesc | NT | P/F | NT | NT | NT | NT | NT | NT | NT |
| I71E | No | No | No | No | 4.0 | 1.1 | <0.01 | 0 | <0.025 | 0.63 | 26.7/6.4 | 4.9 |
| I71R | No | No | No | No | 3.8 | 1.0 | <0.01 | 0 | <0.025 | 0.28 | 25.1/6.3 | 4.5 |
| I71W | No | No | No | NT | 4.5 | 1.1 | <0.01 | 0 | <0.025 | 1.0 | 24.3/6.3 | 4.5 |
| R72E | No | No | Yesc | Yes | 3.6 | 0.59 | NT | NT | NT | 0.49 | 21.8/5.5 | 4.4 |
| S77W | Yes | No | Yes | NT | P/F | NT | NT | NT | NT | NT | NT | NT |
| Y79R | No | No | No | No | 3.3 | 0.42 | 0.053d | 0 | 0.13 | 0.62 | 27.2/6.6 | 5.0 |
| K81A | NT | Yes | Yes | NT | 4.4 | 1.0 | 0.12b | 1.5 | NT | NT | 13.4/4.2 | 8.8 |
| K81E | No | No | No | No | 4.5 | 1.0 | <0.01 | 0 | <0.025 | 0.67 | 25.0/5.8 | 4.5 |
| K81F | NT | Yes | Yes | NT | 4.5 | 0.77 | 0.027b | 4.4 | 0.2 | 0.79 | 13.0/4.4 | 8.3 |
| K81L | NT | Yes | Yes | NT | 4.0 | 0.9 | 0.25b | 6.3 | NT | 1.0 | 13.2/4.4 | 8.9 |
| K81Y | NT | Yes | Yes | NT | 4.5 | 1.0 | 0.018b | 3.7 | NT | 0.35 | 13.2/4.6 | 8.6 |
| K84W | Yes | No | No | NT | P/F | NT | NT | NT | NT | NT | NT | NT |
| G86W | Yes | No | No | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| P107W | No | No | Yesc | NT | 4.2 | 1.1 | 0.04b | 3.3 | 0.5 | 0.39 | 19.9/6.2 | 4.4 |
| P107Y | No | No | Yes | NT | 3.6 | 1.1 | 0.055b | 0.54 | NT | 0.38 | 20.2/5.6 | 8.3 |
| A111E | No | No | Yes | NT | 4.0 | 0.91 | 0.023b | 0.92 | NT | 1.2 | 15.9/4.8 | 6.9 |
| Column | 1 | 2 | 3 | 4 | 5 | 6a | 7a | 8a | 9a | 10 | 11 | 12 |
P/F, Partially folded; N/D, not detectable; NT, not tested.
a
The numerical values for the mutants in column 6–9 are the fractional affinities or activities compared with wild type (e.g. “0.037” indicates that the mutant retains 3.7% of wild type activity).
b
Affinity of wild type (Kd=0.21 μM) and fractional activity of mutants determined by nucleotide exchange.
c
Complements but grows slower; colonies are small.
d
By inhibition of wild type promoted nucleotide exchange.