Trigger factor assisted folding of green fluorescent protein.


Abstract

Guanidine induced equilibrium and kinetic folding of a variant of green fluorescent protein (F99S/M153T/V163A, GFPuv) was studied. Using manual mixing and stopped-flow techniques, we combined different probes, including tryptophan fluorescence, chromophore fluorescence and reactivity with DTNB, to trace the spontaneous and TF-assisted folding of guanidine denatured GFPuv. We found that both unfolding and refolding of GFPuv occurred in a stepwise manner and a stable intermediate was populated under equilibrium conditions. The thermodynamic parameters obtained show that the intermediate state of GFPuv is quite compact compared to the denatured state and most of the green fluorescence is retained in this state. By studying GFPuv folding assisted by TF and a number of TF mutants, we found that wild-type TF catalyzes proline isomerization and accelerates the folding rate at low TF concentrations, but retards GFPuv folding and decelerates the folding rate at high TF concentrations. This reflects the two activities of TF, as an enzyme and as a chaperone. A general mechanism of TF assisted protein folding is discussed. Study holds ProTherm entries: 23279, 23280, 23281, 23282, 23283 Extra Details: Trp fluorescenceb, 1 mM EDTA and 5 mM or 1 mM DTT added in the experiment Green fluorescent protein GFP, Aequorea victoria, Trigger factor, folding, chemical denaturation.

Submission Details

ID: XCgySJ5H

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

Publication Details
Xie JB;Zhou JM,Biochemistry (2008) Trigger factor assisted folding of green fluorescent protein. PMID:18067273
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1B9C 1999-02-09T00:00:00+0000 2.4 Green Fluorescent Protein Mutant F99S, M153T and V163A
1BFP 1997-04-09T00:00:00+0000 2.1 BLUE VARIANT OF GREEN FLUORESCENT PROTEIN
1C4F 1999-08-21T00:00:00+0000 2.25 GREEN FLUORESCENT PROTEIN S65T AT PH 4.6
1CV7 1999-08-23T00:00:00+0000 2.5 Crystal structure of enhanced cyan-emission variant of GFP
1EMA 1996-08-01T00:00:00+0000 1.9 GREEN FLUORESCENT PROTEIN FROM AEQUOREA VICTORIA
1EMB 1997-01-08T00:00:00+0000 2.13 GREEN FLUORESCENT PROTEIN (GFP) FROM AEQUOREA VICTORIA, GLN 80 REPLACED WITH ARG
1EMC 1997-03-31T00:00:00+0000 2.3 GREEN FLUORESCENT PROTEIN FROM AEQUOREA VICTORIA, MUTANT
1EME 1997-03-31T00:00:00+0000 2.5 GREEN FLUORESCENT PROTEIN FROM AEQUOREA VICTORIA, MUTANT
1EMF 1997-03-31T00:00:00+0000 2.4 GREEN FLUORESCENT PROTEIN FROM AEQUOREA VICTORIA, MUTANT
1EMG 1998-11-12T00:00:00+0000 2.0 GREEN FLUORESCENT PROTEIN (65-67 REPLACED BY CRO, S65T SUBSTITUTION, Q80R)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Green fluorescent protein P42212 GFP_AEQVI