Inhibition Of Urea-and Acid-induced Protein Denaturations By Trehalose

Protein stability is significantly pivotal not only for theoretical research inrelation with structure and function of proteins, but also for application in productionof protein pharmaceuticals. Among the polyols, trehalose has been found particularlyefficient in enhancing protein stability. However, molecular mechanism,thermodynamic and kinetic behaviors for inhibition of urea-and acid-induced proteindenaturations by trehalose are still unclear. Based on these problems, counteractingeffects of trehalose on urea-and acid-induced protein unfolding are systematicallyinvestigated using molecular dynamics (MD) simulations combined with a variety ofexperimental techniques in this study.In inhibition of urea-induced protein denaturation by trehalose, all-atom MDsimulations were firstly performed to investigate the molecular mechanism forprotective effects of trehalose against urea-induced protein denaturation. It is foundthat trehalose binds urea via hydrogen bonds, so urea molecules are also expelledfrom the protein surface along with the preferential exclusion of trehalose. Thus,trehalose effectively inhibits the denaturing effects of urea on protein. Then thekinetic behavior for inhibition of urea-induced protein unfolding by trehalose wasstudied by stopped-flow fluorescence spectroscopy. The unfolding pathway is notchanged by trehalose at lower concentrations. However, the rate constant andamplitude for the fast unfolding phase decrease with increasing trehaloseconcentration. At higher trehalose concentrations, the unfolding pathway istransformed from the biphasic process into a single slow phase. The rate constant andamplitude for the single phase also decrease with increasing trehalose concentration.Furthermore, in inhibition of acid-induced protein denaturation by trehalose, aseries of spectroscopic techniques were used to investigate counteraction of trehaloseon acid-induced protein denaturation. It is found that trehalose induceds theacid-induced unfolded protein into a molten globule state. Then stopped-flowfluorescence spectra indicate that trehalose at low concentrations has almost noinfluence on the unfolding transition. However, at higher trehalose concentrations, itis transformed into a biphasic process without the fast phase from the initial triphasicpathway. The rate constant for all the three unfolding phases decreases linearly withincreasing trehalose concentration. Meanwhile, the amplitude for the fast andintermediate phases diminishes with the increment of trehalose concentration. Nevertheless, there is little influence of trehalose on the amplitude in the slow phase.Therefore, trehalose kinetically inhibits acid-induced protein denaturation mainly byrestricting conformational changes in proteins during the unfolding process andslowing down protein unfolding rate.Based on above experiments, synergistical inhibition of acid-induced proteindenaturation by trehalose and NaCl was further studied. Fluorescence spectra andthermodynamic analysis confirm that trehalose and NaCl could synergistically inhibitacid-induced protein unfolding. Besides, it is found that the kinetic pathway ofacid-induced protein unfolding is successively transformed from the initial triphasicpathway into a biphasic process and a single slow phase with increasing NaCl and/ortrehalose concentration by analyzing stopped-flow fluorescence spectra data. Thekinetic behavior of synergistical inhibition of trehalose and NaCl is mainly throughrestricting the conformational expansion of proteins during the unfolding process. Butthere is little influnce on protein unfolding rate.