The effect of crowding agents and salts on the structure and stability of ubiquitin mutant V26A/I30A

• Main Authors: Chien-Ming Lan, 藍健銘
• Other Authors: Chia-Lin Chyan
• Format: Others
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/265y5j

碩士 === 國立東華大學 === 化學系 === 105 === In the first part of this paper, the effects of crowding agents, like pH value, NaCl, and dextran, on the secondary structure of ubiquitin mutant V26A/I30A were studied by circular dichroism spectroscopy. In the second part, the effects of NaCl and dextran concentration on the V26A/I30A were studied by using nuclear magnetic resonance spectroscopy in different pH value. The differences in the structures of ubiquitin mutants V26A/I30A, I30A and wild-type ubiquitin were examined. In the previous study, it has already known that the wild-type ubiquitin maintains folded state in the acidic or neutral environment. In this article, the wild-type ubiquitin was mutated in the hydrophobic center, and studied by nuclear magnetic resonance spectroscopy. We found that the ubiquitin with V26A/I30A mutation had a folded and unfolded state coexisting in the pH 4.0 environment. When the high concentration of NaCl or dextran was added, the unfolded state gradually disappeared. The results of circular dichroism spectroscopy show that the percentage of disordered structure decreases from 37.7 % to 36.4 %, the ratio of -sheet structure is maintained at 37.1 %, and the -helix structure is increased from the 7.9 % to 9.3 % when increasing the concentration of NaCl. Likewise, the percentage of disorder structure from 35.3 % to 31.9 %, -sheet structure from 36.2 % to 39.1 %, and the -helix structure from 8.5 % to 10.9 % when increasing the concentration of dextran. From the NMR spectroscopy, we found that when increase the concentration of NaCl, the crosspeak of the unfolded state will gradually disappear. The structure of the V26A/I30A mutant ubiquitin will fold again due to the exist of anion in the NaCl. When increasing the concentration of dextran, the crosspeak of the unfolded state is disappears, which indicating that the crowded environment formed by dextran leads to the folding of the unfolded ubiquitin mutant V26A/I30A. We have already resolved the structure of the folded ubiquitin mutant V26A/I30A, and compared with the structure, I30A mutant ubiquitin and wild-type ubiquitin that were solved in our previous study. It was found that the -helix in the hydrophobic center will disappear after the I30A and V26A/I30A mutation of ubiquitin. The distance between the-helix and the -sheet was increase from 14.0 Å increased to 16.5 Å, the -helix secondary structure of the ubiquitin mutant V26A/I30A differs from the -helix secondary structure of the wild-type ubiquitin by 28.7 °. Therefore, the structure of the ubiquitin mutant was considered to be looser than that of the wild-type ubiquitin, and the structure of the ubiquitin mutant V26A/I30A was compared with that of the ubiquitin mutant I30A, the secondary structure of the original ubiquitin mutant I30A part -sheet disappears, the distance between the secondary structure of -helix and the secondary structure of -sheet increases from the original 13.4 Å to 16.5 Å, the -helix secondary structure of the ubiquitin mutant V26A/I30A differs from the -helix secondary structure of the wild-type ubiquitin 25.7°, so the structure of the ubiquitin mutant strain V26A/I30A was considered to be looser than the ubiquitin mutant I30.