Complementation and reconstitution of fluorescence from circularly permuted and truncated green fluorescent protein.
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 beta-strands from its beta-barrel structure, and in two of the variants, adding the omitted peptide sequence in trans reconstitutes fluorescence. Detailed biochemical analysis indicates that GFP with beta-strand 7 "left out" (t7SPm) exists in a partially unfolded state. The apo form t7SPm binds the free beta-strand 7 peptide with a dissociation constant of approximately 0.5 microM 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.
Huang
Yao-ming
YM
Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180-3590, USA.
Bystroff
Christopher
C
D015374:Biosensing Techniques; D018360:Crystallography, X-Ray; D005453:Fluorescence; D005470:Fluorometry; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion; D013056:Spectrophotometry, Ultraviolet
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins