| Autor: |
Melnik BS; Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Russia.; Pushchino Branch, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Russia., Katina NS; Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Russia., Ryabova NA; Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Russia., Marchenkov VV; Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Russia., Melnik TN; Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Russia., Karuzina NE; Biophysics Department, Siberian Federal University, 660041 Krasnoyarsk, Russia., Nemtseva EV; Biophysics Department, Siberian Federal University, 660041 Krasnoyarsk, Russia.; Institute of Biophysics, Siberian Branch of Russian Academy of Sciences, 660036 Krasnoyarsk, Russia. |
| Abstrakt: |
Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A). |
