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Structure and functional complementation of engineered fragments from yeast phosphoglycerate kinase
F Pecorari1, P Minard, M Desmadril
1Laboratoire d'Enzymologie physicochimique et moléculaire, Université de Paris-Sud, Orsay, France.
Protein Engineering
|April 1, 1993
Summary
Yeast phosphoglycerate kinase fragments, unlike structural domains, can reassociate and regain partial function. Correct folding of individual fragments is not essential for this functional complementation.
Area of Science:
- Biochemistry
- Protein folding
- Enzyme kinetics
Background:
- Isolated structural domains of yeast phosphoglycerate kinase refold but do not reassociate functionally.
- Understanding protein fragment reassociation is key to enzyme function restoration.
Purpose of the Study:
- Investigate folding of non-domain complementary protein fragments.
- Determine requirements for fragment reassociation and functional complementation.
Main Methods:
- Site-specific protein cleavage using 5,5'-dithiobis(2-nitrobenzoate)/potassium cyanide.
- Generation of two complementary fragment pairs: 1-96/97-415 and 1-248/249-415.
- Analysis of fragment structure, stability, and functional complementation.
Main Results:
- Short fragments (1-96, 249-415) showed low stability; larger fragments retained high structural content and stability.
- Both fragment pairs exhibited weak but significant complementation, with 1-248/249-415 recovering 8% native enzyme activity.
- Independent refolding affected complementation yield differently for the two fragment pairs.
Conclusions:
- Complementary protein fragments can reassociate and regain enzymatic activity.
- Correct folding of isolated fragments is not a prerequisite for functional complementation.
- Specific fragment pairs demonstrate varying requirements for reassociation and functional recovery.