Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels.


Abstract

Many of the effects exerted on protein structure, stability, and dynamics by molecular crowding and confinement in the cellular environment can be mimicked by encapsulation in polymeric matrices. We have compared the stability and unfolding kinetics of a highly fluorescent mutant of Green Fluorescent Protein, GFPmut2, in solution and in wet, nanoporous silica gels. In the absence of denaturant, encapsulation does not induce any observable change in the circular dichroism and fluorescence emission spectra of GFPmut2. In solution, the unfolding induced by guanidinium chloride is well described by a thermodynamic and kinetic two-state process. In the gel, biphasic unfolding kinetics reveal that at least two alternative conformations of the native protein are significantly populated. The relative rates for the unfolding of each conformer differ by almost two orders of magnitude. The slower rate, once extrapolated to native solvent conditions, superimposes to that of the single unfolding phase observed in solution. Differences in the dependence on denaturant concentration are consistent with restrictions opposed by the gel to possibly expanded transition states and to the conformational entropy of the denatured ensemble. The observed behavior highlights the significance of investigating protein function and stability in different environments to uncover structural and dynamic properties that can escape detection in dilute solution, but might be relevant for proteins in vivo. Study holds ProTherm entries: 19407, 19408 Extra Details: molecular crowding; protein folding; protein immobilization; encapsulation; fluorescence

Submission Details

ID: fEodUHJD

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Campanini B;Bologna S;Cannone F;Chirico G;Mozzarelli A;Bettati S,Protein Sci. (2005) Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels. PMID:15802645
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