Investigating Protein Stability through SUPREX and CD Techniques in Various Variants
This study analyzes the stability of proteins using the SUPREX (Stability of Ubiquitous Proteins from Experiment) and Circular Dichroism (CD) methods, detailing the Gibbs free energy (Gf) of different protein variants. Notable results include variants with mutations such as A66G and Q33Y demonstrating varying stabilities. Measurements were conducted at specific temperatures, with calculations based on known dissociation constants. This research provides insights into how mutations affect protein stability, critical for applications in biochemistry and molecular biology.
Investigating Protein Stability through SUPREX and CD Techniques in Various Variants
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Presentation Transcript
Amide Proton Exchange Protection Factor F = kex,bound / kex,free
SUPREX Ghaemmaghami et al. PNAS 97: 8296 (2000)
Table 1. Stability of proteins by SUPREX and CD denaturation Protein Gf, kcal/mol Gf, kcal/mol SUPREXa CD SUPREXa CD 6-85 5.0 ± 0.4 4.4 ± 0.2b 6-85 (A66G) >4.0 >1.0 1.5c 6-85 (A63G) 4.8 ± 0.4 0.2 0.4c 6-85 (G46A/G48A/A66G) 5.2 ± 0.4 0.2 0.4c 6-85 (G46A/G48A/A49G) 5.6 ± 0.4 0.6 1.0c 6-85 (Q33Y) 6.5 ± 0.4 1.5 1.5d 6-85 (G46A/G48A) 6.7 ± 0.4 6.1 ± 0.2b 1.7 1.7 6-85 (G46A/G48A/Q33Y) 7.9 ± 0.7 2.9 3.4d MBP 16 ± 3 14.5 ± 0.4e 12.5 ± 0.2f MBP + 100 µM maltose 19 ± 3 3.0 2.9g a Stabilities measured at 23°C. b Stabilities measured at 25°C. c Gf (Gfvariant-Gfwild type) were measured at 37°C (23). d Gf measured at 25°C by G. Kapp, personal communication. e Stability measured at 25°C by urea titration (17). f Stability measured at 25°C by GdmCl titration (17). g A dissociation constant of 1 µM (20) and an estimated protein concentration of 5 µM were used to calculate the expected change in stability (3).
