| Co-Investigator(Kenkyū-buntansha) |
TOKUNAGA Fumio Osaka University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (80025452)
MIHARA Ken'ichi Nara Institute of Science and Technology, Graduate School of Materials Science, Lecturer, 物質創成科学研究科, 助手 (10304164)
IMAMOTO Ysushi Nara Institute of Science and Technology, Graduate School of Materials Science, Associate Professor, 物質創成科学研究科, 助教授 (80263200)
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| Research Abstract |
The tertiary fold of the internal deletion mutant, D44-49, of Staphylococcal nuclease (SNase) is identical to that of wild type, but D44-49 is more stable against heat and acid than wild type. D114-119 forms a dimmer with domain swapping. The overall fold of D114-119 is quite similar to that of wild type but is less stable than wild type. Both deletion mutants show now enzymatic activity. D114-119 lacks a substrate-binding ability, while D44-49 can bind a substrate. D44-49 lacks a catalytic activity. Based on the crystal structure analysis of D44-49, it is clarified that the loop flexibility is essential for the catalytic activity.
Systematic deletions from the C terminus of SNase were performed to reveal the role of C-terminal region in folding and functioning. D140-149 is in compact denatured structure, which is identical to D137-149. D140-149 shows enzymatic activity as well as D137-149, and these two mutants possess the substrate-induced folding ability. D142-149 and D143-149 are id
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entical to wild type. D141-149 shows intermediate properties of wild type and D140-149. D141-149/W140A is indistinguishable with D140-149. On the other hand, the substitutions of S141 into A or N show no effects on the structure and activity of D142-149. We can conclude the followings : 1) W140 is a key residue to maintain the native conformation stably under a physiological condition ; 2) the side chain of the 141th residue is not important but main chain information is important to take a native conformation ; 3) if the polypeptide shows an enzymatic activity, the polypeptide can fold into a native conformation, but the foldability is not necessarily related to the activity.
In the case of photoactive yeIIow protein, V122 is the essential residue to form holo-protein and to keep native structure stably. If the polypeptide contains up to K123, the polypeptide can behave as PYP in terms of structure and function. R124 makes photoreaction efficient. V125 makes native structure stable.
These results also suggest that the folding in sequence space is two-state transition. Less
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