- 作者: 顏克明, 黃秀慧, 黃福永
- 作者服務機構: Department of Chemistry, National Cheng Kung University, Tainan 70101, Taiwan, R.O.C.
- 中文摘要: In order to explore whether hydrophobicity or charge at the 71th residue is important for chaperone-like activity, wild type (wt) and two mutantαA-crystallins, F71W and F71R, with an extra 11 residues including 6 histidines tagged at the N-terminal were prepared. Chaperone-like activity toward dithiotreitol (DTT) induced insulin aggregation showed the 11 extra residues had little effect. The activity for F71W was as active as the wild type, while F71R was much less active. For the wild type and F71W, the activity increased with the increase of molar quantity; surprisingly, for F71R it caused a drastic decline. ANS fluorescence measurements showed F71R had the highest surface hydrophobicity, suggesting positive charge at this site caused the conformational alteration leading to the increase of hydrophobicity and this hydrophobicity resulted in the formation of high molecular weight aggregates (> 2000 kDa) observed from the thermal stability study. The thermal stability study revealed F71W was the most thermally stable crystallin. Chaperone-like activity studies at various temperatures suggested the monomer and oligomer, 650 kDa, were responsible for chaperone function and surface hydrophobicity was not the prerequisite for activity. Near-UV CD studies showedβ-sheet was the major secondary structure for all three crystallins and F71R had a higher percentage of random coil.
- 英文摘要: --
- 中文關鍵字: Site-directed mutagenesis; aA-crystallin; Structural change; Chaperone activity; Circular dichroism; Hydrophobicity; Oligomerization; ANS fluorescence.
- 英文關鍵字: --