Table 1.
Dissociation constants of complexes between pGolemi variants and peptides and Mena1-112, VASP1-115, or EVL1-115 at 25°C. Also shown is the mean residue ellipticity at 222 nm of each variant in the absence of EVH1 domain (10 mM NaH2PO4 100 mM NaCl (pH 7.5) at 25°C). Colors identify the location of alanine substitution: red and pink identify variants with substitution of an ActA-derived residue, whereas light blue and dark blue identify variants with substitution of an aPP-derived folding residue. Values in brackets indicate Kd values estimated from ill-behaved binding isotherms. N.D. – Not Determined, N.C. – Not Calculated. Two pGolemi variants, P7A and F19A, reproducibly showed ill-behaved tryptophan perturbation profiles, perhaps due to aggregation, and were not included in further analysis.
|
Kd Mena (μM) |
ΔGMena (kcal• |
Kd VASP (μM) |
ΔGVASP (kcal•mol− |
ΔΔGsp Mena-VASP |
Kd Evl (μM) |
ΔGEvl (kcal• |
ΔΔGsp Mena-Evl | MRE222 (deg•cm2• |
|
|---|---|---|---|---|---|---|---|---|---|
| ActA11 | 6 ± 2 | −7 | 15 ± 4 | − 6.6 | −0.5 | 4.1 ± 0.3 | −7.3 | 0.23 | N.D. |
| pGolemi | 0.6 ± 0.2 | − 8.5 | 13 ± 2 | − 6.7 | −1.8 | >100 | > −5.5 | < − 3.1 | −16262 |
| pGol-2 | >100 | > −5.5 | 15 ± 5 | − 6.6 | <1.1 | 10± 2 | −6.8 | < 1.3 | −20386 |
| P1A | 0.8 ± 0.3 | −8.3 | 5.4 ± 0.1 | − 7.2 | −1.1 | 20 ± 2 | −6.4 | −1.9 | −5522 |
| P1A11 | > 480 | > − 4.5 | >240 | > − 5.3 | N.C. | >240 | > −4.9 | N.C. | N.D. |
| F2A | 55 ± 6 | − 5.8 | 56 ± 3 | − 5.8 | 0.0 | 106 ± 10 | −5.4 | −0.4 | −13745 |
| F2L | 1.0 ± 0.1 | −8.2 | 8.0 ± 0.4 | − 6.9 | −1.3 | 23 ± 1 | −6.3 | −1.9 | −15052 |
| P3A | 2.3 ± 0.5 | −7.7 | [>75] | [> − 5.6] | N.C. | [> 50] | [> −5.9] | N.C. | −10745 |
| P3Z | 2.5 ± 0.3 | −7.6 | 7 ± 1 | − 7.0 | − 0.6 | > 50 | > −5.9 | < − 1.8 | −9384 |
| P4A | 3.3 ±0.4 | −7.5 | 4 ± 1 | − 7.3 | − 0.2 | 43 ± 5 | − 6.0 | −1.5 | −8205 |
| P4Z | 2.4 ± 0.4 | −7.7 | 5 ± 1 | − 7.2 | − 0.5 | > 50 | > − 5.9 | < − 1.8 | −5384 |
| T5A | 2.2 ± 0.2 | −7.7 | 1.3 ± 0.2 | − 8.0 | 0.3 | 13 ± 2 | − 6.7 | − 1.0 | −8667 |
| T5A11 | > 800 | > − 4.2 | > 240 | > − 5.3 | N.C. | 210 ± 30 | − 5.0 | > 0.78 | N.D |
| T5L | 1.5 ± 1 | − 7.9 | 10 ± 1 | − 6.8 | − 1.1 | 22 ± 1 | − 6.4 | − 1.5 | −14647 |
| P6A | 9 ± 2 | − 6.9 | 34 ± 3 | − 6.1 | − 0.8 | [> 50] | [> − 5.9] | N.C. | −5937 |
| P7A | [> 50] | [> − 5.9] | [> 100] | [> − 5.5] | N.C. | [> 100] | [> − 5.5] | N.C. | −2932 |
| E9A | 0.9 ± 0.2 | − 8.2 | 8 ± 1 | − 6.9 | − 1.3 | 36 ± 3 | − 6.1 | − 2.2 | −13115 |
| E10A | 1.2 ± 0.2 | − 8.1 | 2.3 ± 0.3 | − 7.7 | − 0.4 | 32 ± 2 | − 6.1 | − 1.9 | −8261 |
| A11L | 12 ± 2 | − 6.7 | 8 ± 4 | − 6.9 | 0.2 | 23 ± 10 | − 6.3 | − 0.4 | − 12893 |
| L16A | 14 ± 3 | − 6.6 | > 50 | > − 5.9 | < − 0.7 | >50 | > − 5.9 | < −0.7 | −6714 |
| F19A | [> 100] | [> − 5.5] | [> 100] | [> − 5.5] | N.C. | [>50] | [> − 5.9] | N.C. | −5756 |
| L23A | 77 ± 8 | −5.6 | > 50 | > − 5.9 | < 0.3 | >50 | > − 5.9 | < 0.3 | −4945 |
| Y26A | 3.0 ± 0.3 | −7.5 | > 50 | > − 5.9 | < − 1.7 | >150 | > − 5.9 | < − 2.3 | −7234 |
| V29A | 2.6 ± 0.5 | −7.6 | [> 50] | [> − 5.9] | N.C. | [>100] | [> − 5.9] | N.C. | −11797 |