Table 2 |
||||||
|
SEC and SPR evaluation of predicted significantly destabilized MICA point mutants |
||||||
|
Buffer + 0 M Urea |
||||||
|
Elution |
Apparent |
Apparent |
||||
|
MIC Mutant |
Design Score |
ΔGeq (kcal mol-1) |
SEC Binding |
Volume (mL) |
MW (kDa) |
r (Å) |
|
|
||||||
|
WT |
0 |
-7.3 ± 0.1 |
+ |
3.5 ± 0.1 |
30 ± 2 |
23 ± 2 |
|
N69W_K152E_K154D |
4 |
-8.8 ± 0.1 |
++ |
3.4 ± 0.1 |
30 ± 2 |
23 ± 2 |
|
Y157W |
50 |
-7.9 ± 0.1 |
- |
3.6 ± 0.1 |
30 ± 2 |
23 ± 2 |
|
K154P |
220 |
-7.6 ± 0.1 |
+ |
3.1 ± 0.1 |
34 ± 2 |
25 ± 2 |
|
Q108W |
290 |
-7.8 ± 0.2 |
+ |
3.1 ± 0.1 |
34 ± 2 |
25 ± 2 |
|
|
||||||
|
Buffer + 1 M Urea |
||||||
|
Elution |
Apparent |
Apparent |
||||
|
MIC Mutant |
SEC Binding |
Volume (mL) |
MW (kDa) |
r (Å) |
||
|
|
||||||
|
WT |
- |
2.8 ± 0.1 |
38 ± 2 |
27 ± 3 |
||
|
N69W_K152E_K154D |
++ |
2.9 ± 0.1 |
37 ± 2 |
26 ± 3 |
||
|
Y157W |
- |
2.8 ± 0.1 |
39 ± 2 |
27 ± 3 |
||
|
K154P |
- |
2.6 ± 0.1 |
41 ± 3 |
28 ± 3 |
||
|
Q108W |
- |
2.7 ± 0.1 |
40 ± 3 |
28 ± 3 |
||
|
|
||||||
|
Design scores are listed relative to wild-type. ΔG values determined at equilibrium by SPR. Wild-type and high-affinity MIC N69W_K152E_K154D SPR values as reported previously in [13]. Upper table shows SEC results with no added urea, and lower table shows SEC results in the presence of 1 M additional urea. Results of SEC binding assay follow the examples in Figure 2. Elution volume measured relative to the elution time of blue dextran. Apparent molecular weight (MW) and radii (r) determined by line fit of elution time to Low Molecular Weight Standards (GE Healthcare). Standard errors are from triplicate experiments as described in Methods. |
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|
Mayer et al. BMC Research Notes 2009 2:135 doi:10.1186/1756-0500-2-135 |
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