Role of histidine 148 in stability and dynamics of a highly fluorescent GFP variant.
The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.
Dipartimento di Farmacia, Università degli Studi di Parma, Parma, Italy. barbara.campanini@unipr.it
Barbara
B
Campanini
Barbara
B
Pioselli
Samanta
S
Raboni
Paolo
P
Felici
Immacolata
I
Giordano
Laura
L
D'Alfonso
Maddalena
M
Collini
Giuseppe
G
Chirico
Stefano
S
Bettati
D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D049452:Green Fluorescent Proteins; D006639:Histidine