title: Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel beta strands of green fluorescent protein.
abstract: We have identified pairs of residues across the two parallel beta strands of green fluorescent protein that facilitate native strand register of the surface-exposed beta barrel. After constructing a suitable host environment around two guest residues, minimizing interactions of the guest residues with surrounding side-chains yet maintaining the wild-type protein structure and the chromophore environment, we introduced a library of cross-strand pairings by cassette mutagenesis. Colonies of Escherichia coli transformed with the library differ in intracellular fluorescence. Most of the fluorescent pairs have predominantly charged and polar guest site residues. The magnitude and the rate of fluorescence acquisition in vivo from transformed E. coli cells varies among the mutants despite comparable levels of protein expression. Spectroscopic measurements of purified mutants show that the native protein structure is maintained. Kinetic studies using purified protein with fully matured chromophores demonstrate that the mutants span a 10-fold range in folding rates with undetectable differences in unfolding rates. Thus, green fluorescent protein provides an ideal system for monitoring determinants of in vivo protein folding. Cross-strand pairings affect both protein stability and folding kinetics by favoring the formation of native strand register preferentially to non-native strand alignments.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Merkel
authors.forename: L
authors.initials: L
authors.lastname: Regan
mesh_terms: D000818:Animals; D004926:Escherichia coli; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D017510:Protein Folding; D013057:Spectrum Analysis
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins