Denaturant-dependent folding of GFP

Abstract

We use molecular simulations using a coarse-grained model to map the folding landscape of Green Fluorescent Protein (GFP), which is extensively used as a marker in cell biology and biotechnology. Thermal and Guanidinium chloride (GdmCl) induced unfolding of a variant of GFP, without the chromophore, occurs in an apparent two-state manner. The calculated midpoint of the equilibrium folding in GdmCl, taken into account using the Molecular Transfer Model (MTM), is in excellent agreement with the experiments. The melting temperatures decrease linearly as the concentrations of GdmCl and urea are increased. The structural features of rarely populated equilibrium intermediates, visible only in free energy profiles projected along a few order parameters, are remarkably similar to those identified in a number of ensemble experiments in GFP with the chromophore. The excellent agreement between simulations and experiments show that the equilibrium intermediates are stabilized by the chromophore. Folding kinetics, upon temperature quench, show that GFP first collapses and populates an ensemble of compact structures. Despite the seeming simplicity of the equilibrium folding, flux to the native state flows through multiple channels and can be described by the kinetic partitioning mechanism. Detailed analysis of the folding trajectories show that both equilibrium and several kinetic intermediates, including misfolded structures, are sampled during folding. Interestingly, the intermediates characterized in the simulations coincide with those identified in single molecule pulling experiments. Our predictions, amenable to experimental tests, show that MTM is a practical way to simulate the effect of denaturants on the folding of large proteins.complex folding landscape of GFP | equilibrium and kinetic pathways | multiple folding routes | self-organized polymer model | multicanonical simulation O ur understanding of the folding mechanisms of small single domain proteins has advanced significantly over the last twenty years thanks to theoretical developments (1-7), simulations (8-13), and advances in experimental methods (14-19). Indeed, it can be justly stated that simulations of structure-based models are remarkably successful in predicting the folding mechanisms in the presence (20-22) and absence of denaturants (23-25). More refined questions, such as the relationship between pathway diversity and the symmetry of the underlying native structures (26), transition path times for crossing free energy barriers (27), the nature of the transition state ensemble (28-31) have also been addressed using theory and experiments. In contrast, much less is known about how large proteins with number, N, of amino acids exceeding ≈200 with complex β-sheet fold.Folding mechanisms of Green Fluorescent Protein (GFP), with predominantly β-sheet native structures stabilized by contacts between residues that are well separated along the sequence, have been extensively investigated (32-40). However, the details of the folding kinetics including the st...

Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK

“…The resulting computational predictions have been confirmed by our joint studies combining simulation and atomic force microscopy experiments (28). Importantly, this model, which enables one to follow the dynamics of proteins on experimental timescales up to seconds (29), has been successfully applied to study the mechanical response of systems from kinesin (30) to fibrinogen (31) to viral capsids (32) to full microtubules (33) as well as to provide insights into protein refolding following chemical denaturation (34,35).…”

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

“…(1,2,14). It is known that fluorescence is quenched upon denaturation and that several intermediates have been observed in folding experiments and simulations (3,(15)(16)(17)(18)(19)(20)(21), although it has not been possible to definitively probe the fluorescence properties of partially structured intermediates in isolation from the native state. Speculation exists regarding whether single-molecule photobleaching can be reversed by unfolding and refolding the protein (22).…”

Knotting and unknotting of a protein in single molecule experiments

“…Extensive experimental and computational studies have provided significant mechanistic insight into folding pathways (1)(2)(3)(4)(5). These proteins, which fold rapidly, have been shown to possess relatively smooth energy landscapes (6,7).…”