Sequence and Misfolding in the Ancient TIM Barrel Motif

Abstract

Protein folding energy landscapes have been driven by evolution to minimize the energetic and topological frustration experienced during the folding reaction. Small 2 state folders, whose folding rates correlate with their topologies, are good examples of evolution at work. However, larger proteins are often found to have partially-folded intermediates on their energy landscape, some of which can be misfolded. (βα)8 TIM barrel proteins, one of the most common motifs in biology, are one such class of proteins. Previous studies on several diverse TIM barrels have shown that the structures of their misfolded intermediates are not conserved. We hypothesize that large, local in sequence clusters of isoleucine, leucine, and valine (ILV) residues drive the formation of the misfolded intermediate in TIM barrels. These clusters are not conserved in the structures, but do correlate with strong protection against hydrogen exchange in folding intermediates. To test the generality of this hypothesis, we expanded our studies to the indole-3-glycerol phosphate synthase from S. solfataricus (sIGPS). sIGPS has two ILV clusters in different locations than those found in the E. coli α-subunit of trp synthase. The common fold but sequence variations allows a test of the importance of ILV clusters in the early misfolding events. Continuous-flow trFRET has been used to measure pair-wise distance distributions for several βα segments in the misfolded intermediate that appears after a few 10's of microseconds. To complement the pair-wise measurements, SAXS experiments provide global dimensions of the earliest folding events. The insights obtained will explore the link between sequence, ILV clusters and misfolding for the TIM barrel architecture. NIH GM23303.