Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel beta strands of green fluorescent protein

We have identified pairs of residues across the two parallel beta strands o f green fluorescent protein that facilitate native strand register of the s urface-exposed beta barrel. After constructing a suitable host environment around two guest residues, minimizing interactions of the guest residues wi th surrounding side-chains yet maintaining the wild-type protein structure and the chromophore environment, we introduced a library of cross-strand pa irings by cassette mutagenesis. Colonies of Escherichia coli transformed wi th the library differ in intracellular fluorescence. Most of the fluorescen t pairs have predominantly charged and polar guest site residues. The magni tude and the rate of fluorescence acquisition in vivo from transformed E. c oli cells varies among the mutants despite comparable levels of protein exp ression. Spectroscopic measurements of purified mutants show that the nativ e protein structure is maintained. Kinetic studies using purified protein w ith fully matured chromophores demonstrate that the mutants span a 10-fold range in folding rates with undetectable differences in unfolding rates. Th us, green fluorescent protein provides an ideal system for monitoring deter minants of in vivo protein folding. Cross-strand pairings affect both prote in stability and folding kinetics by favoring the formation of native stran d register preferentially to non-native strand alignments.