OFF-PATHWAY MISFOLDED FOLDING INTERMEDIATES OF CHEY AND VARIANTS

Illinois Institute Of Technology, Chicago IL

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal is part of an overall goal of determining the mechanism by which the amino acid sequences and motifs of the alpha/beta/alpha class of proteins direct their acquisition of secondary and tertiary structure during folding. The folding mechanism of flavodoxin fold proteins has been well studied by several groups. An interesting feature of their folding energy landscape is that an off-pathway folding intermediate has been observed in kinetic refolding experiments for all flavodoxin fold proteins, including CheY. In a recent publication we have demonstrated experimentally and computationally that the off-pathway species is an obligate intermediate that forms within the dead time of stopped-flow instrumentation. The intermediate has considerable secondary structure and stability and remains populated for ~100 s. Go-modeling results from our collaborators and hydrogen-exchange mass spectrometry data (unpublished results) suggest that the source of this energetic frustration is the early formation of contacts between the N- and C- terminal halves of the protein (between a4 and a3 and between b4 and b3) which result in a non-productive energetic trap that must be undone in order for the productive transition state intermediate to form. Mutagenesis studies are underway in our lab to replace hydrophobic residues in the a4 and b4 regions and probe their effect on the secondary structure and packing of residues. The equilibrium and time-resolved SAXS will complement these studies by monitoring the resulting changes in the size and shape of the obligate off-pathway intermediate.