NDLI: Complementation and Reconstitution of Fluorescence from Circularly Permuted and Truncated Green Fluorescent Protein †

Content Provider	PubMed Central
Author	Huang, Yao-ming Christopher, Bystroff
Abstract	Green fluorescent protein (GFP) has been used as a proof of concept for a novel “leave-one-out” biosensor design in which a protein that has a segment omitted from the middle of the sequence by circular permutation and truncation binds the missing peptide and reconstitutes its function. Three variants of GFP have been synthesized that are each missing one of the 11 β-strands from its β-barrel structure, and in two of the variants, adding the omitted peptide sequence in trans reconstitutes fluorescence. Detailed biochemical analysis indicates that GFP with β-strand 7 “left out” (t7SPm) exists in a partially unfolded state. The apo form t7SPm binds the free β-strand 7 peptide with a dissociation constant of ~0.5 µM and folds into the native state of GFP, resulting in fluorescence recovery. Folding of t7SPm, both with and without the peptide ligand, is at least a three-state process and has a rate comparable to that of the full-length and unpermuted GFP. The conserved kinetic properties strongly suggest that the rate-limiting steps in the folding pathway have not been altered by circular permutation and truncation in t7SPm. This study shows that structural and functional reconstitution of GFP can occur with a segment omitted from the middle of the chain, and that the unbound form is in a partially unfolded state.
Related Links	http://dx.doi.org/10.1021/bi802027g
Ending Page	940
Page Count	12
Starting Page	929
File Format	PDF
ISSN	00062960
e-ISSN	15204995
Journal	Biochemistry
Issue Number	5
Volume Number	48
Language	English
Publisher Date	2009-02-10
Access Restriction	Open
Subject Keyword	Biochemistry Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biochemistry

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