Thermal unfolding wild-type protein

Although methionine is present with relatively low frequency in naturally-occurring proteins (Klapper, 1977), in this case it appears to act as a normal hydrophobic residue. The thermal denaturations of all single variants are essentially as cooperative as wild-type with comparable enthalpies of unfolding (Table II). This is also the case for the seven- but less so for the ten-methionine variant. 

Here, and k2 are the unfolding rate constants for the unfolding transition from N to I and from I to respectively. The unfolding kinetics can be quantitatively analyzed from the scanned intensities of the Coomassie blue stained tailspike bands on SDS gels. Figure 1 depicts results from a typical thermal unfolding experiment for wild type tailspike protein, which was performed in Tris buffer 8) and 2% SDS at 65 C. Kinetic analysis yields two rate constants 1.1 x 10 s and 4.0 x 10 s for the conversion from N to I and from I to A/, respectively. 

Figure 1. Thermal unfolding of wild type tailspike protein at 65 C. Thermal unfolding was performed by incubating 0.4 mg/ml tailspike prepared in 50 mM Tris (pH 8), 1.7 mM 2-mercaptoethanol and 2% SDS at 65°C. Samples were taken at the indicated times. The reaction was quenched by mixing the samples with SDS sample buffer (62.5 mM Tris at pH 7, 2.1 mM 2-mercaptoethanol, 10% glycerol, 0.012% Bromophenol blue dye and 2% SDS) in the cold. Then the samples were electrophoresed through SDS-PAGE at about 20°C and the proteins were stained with Coomassie blue.

Figure 2. Dependence of unfolding rate constants on pH. Wild type tailspike protein was prepared in 50 mM Tris, 1.7 mM 2-mercaptoethanol and 2% SDS and adjust to different pH values by 1 N HCl. Thermal unfolding was done at 65°C and followed by SDS-PAGE at about 20°C. Sample pH values shown here have been corrected to 65°C. kj (a) and k2 ( ) shown in log are the thermal unfolding rate constants for the conversions from N to I and from I to M, respectively. The linear lines through the data points are the results of least-square fit to each individual pH phase for both kj and k2 data. The calculated slopes of the fitting lines for kj are -0.46 and 0.35 for the low and high pH phases, respectively and for k2 are -1.9 and 1.1 for the low and high pH phases, respectively.

Bamase, like CI2, has been the subject of extensive studies, using the protein engineering method combined with kinetic and equilibrium experiments, to investigate its fold-ing/unfolding pathway." Molecular dynamics simulations of thermal denaturation of the wild-type and two mutant proteins (Tyr 17 —> Gly and He 88 —> Val) have been performed to investigate the effect of mutations on the unfolding pathway and transition state." ... 

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