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Table 2 SEC and SPR evaluation of predicted significantly destabilized MICA point mutants

From: Size-exclusion chromatography can identify faster-associating protein complexes and evaluate design strategies

    

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

  1. 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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BMC Research Notes

ISSN: 1756-0500

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