Research Projects

The recent discovery of "metamorphic" proteins with more than one unique structure (and/or function) of comparable stabilities has challenged Anfinsen's one sequence-one structure paradigm. Bioinformatics analyses have predicted that 4% of the proteome are metamorphic and either haven’t been discovered or have eluded traditional structure determination efforts which tend to capture a “static snapshot” or the maximally populated species. Nevertheless, at least 20 metamorphic proteins have been discovered experimentally till date and studied using a variety of biophysical techniques. RfaH is a multi-domain fold switching protein belonging to the NusG family of transcription factors in bacteria. It is the only protein in the family where the C terminal domain (CTD) can exist either in an α-helical hairpin conformation in the closed autoinhibited state of the full length (FL) protein or in a β-barrel conformation in isolation. RfaH controls the expression of long difficult-to-express genes with a promoter proximal 12 nt “ops” sequence, often associated with bacterial virulence. Interestingly, the specificity for ops-containing DNA sequences appears to be an allosteric effect: the isolated NTD acts a transcriptional regulator for all genes, much like the housekeeping factor NusG. The negligible stability of α-helical hairpin conformation in FL RfaH in isolation along with the high co-operativity of unfolding of the FL protein (data from our lab) suggests that transient interdomain interactions in the unfolded ensemble might drive folding. Furthermore, we hypothesize that the 14-residue linker connecting the NTD and CTD domains is responsible for maintaining a high effective concentration (Ceff) of the CTD and increasing the interdomain affinity (Kintra). The linker sequence is not conserved in the NusG family, with a variety of multidomain topologies adopted by its various members. This proposal aims to probe the interdomain interactions in FL RfaH in the unfolded state and modulate the interdomain affinity to understand the origins of ops-specificity. Briefly, the 14 aa WT linker will be replaced by flexible linkers of varying lengths. The resulting constructs will be expressed, purified and characterized using fluorescence, CD and NMR spectroscopy. Ceff will be calculated using polymer physics models and compared with experimental data, determined from competition experiments with isolated CTD. We hypothesize that sufficiently high concentrations of isolated CTD will replace the bound CTD in WT RfaH, which will be probed using tryptophan fluorescence and 19F NMR. By a judicious placement of spectroscopic probes in the WT and CTD RfaH, the shift in the equilibria between open and closed forms of RfaH with increasing concentrations of titrant will be monitored. The outcome of this research will provide fundamental insight into how fold switching can be modulated by domain interactions and test the basis of ops specificity in RfaH.