Native denaturation differential scanning fluorimetry: Determining the effect of urea using a quantitative real-time thermocycler.
The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding (Knd), and protein unfolding by urea (I50). This demonstrates that DSF methods can determine the structural stability of an enzyme's active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity.
Carleton University, Department of Biology, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada.
Christine L
CL
Childers
Carleton University, Department of Biology, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada.
Stuart R
SR
Green
Carleton University, Department of Biology, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada.
Neal J
NJ
Dawson
Carleton University, Department of Biology, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada. Electronic address: kenneth_storey@carleton.ca.
Kenneth B
KB
Storey
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D005470:Fluorometry; D011489:Protein Denaturation; D013997:Time Factors; D014508:Urea / Q000032:analysis* / Q000737:chemistry
D014508:Urea
Differential scanning fluorimetry; Enzyme kinetics; Enzyme stability; Lactate dehydrogenase; Native denaturation; Urea