Determination energy level of intermediate and transition state of Mnemiopsin

Introduction: Mnemiopsin 1 is a member of photoprotein family, made up of 206 amino acid residues. We examined whether three mutations, namely R39K, S128G and V183T affect the thermodynamic stability. Protein engineering analysis with stopped-flow fluorescence measurements and chevron plot analysis may also be used to characterize transition states and intermediate structures of refolding reaction. Materials and methods: Kinetic traces were analyzed by fitting to exponential function of equation using Biokine software: F(t)= at+b+ni Ci exp(kit) Where; F(t) is the fluorescence signal at time t, Ci is the amplitude, ki the rate constant, a the slope of the drift and b is the offset of kinetic curve corresponding to the baseline. Results: In the first model of analysis, we assumed two-state folding. It is evident that refolding arm of the chevron plot shows a deviation from linearity which becomes evident at low urea concentrations, showing a clear systematic deviation from the expected values for a two-state behavior. The low quality of fitting to a two state model indicates that chevron plots should be fitted to other models rather than two state one. In three-state model, we assumed that the chevron plot curvature at low urea concentrations indicates the transient accumulation of a partially folded species with a partially non-native topology, which is less sensitive to urea. Conclusion: According to resulting data and comparison of equilibrium constants for formation of intermediate state from unfolded structure (KUI), it reveals that accumulation of intermediate state during refolding reaction is greater in R39K and V183T relative to the WT and S128G variants. The formation of native structure from intermediate state (kIN) is accompanied by a further molecular rearrangement of molecule and the values of rate constants of reverse reaction (kNI) are negligible when compared with those of forward reaction (kIN).

کلیدواژه ها:

Mnemiopsin ، Chevron plot ، Urea-induced unfolding ، Stopped flow ، Intermediate