Folding Studies of Beta-Strand-Containing Repeat Proteins Through Naturally-Occurring and Consensus-Designed Sequences

Abstract

Repeat proteins, devoid of sequence-distant contacts observed in globular proteins, are ideal candidates for the dissection of local stability, nearest-neighbor contact parameters, cooperativity, and determination of folding energy landscapes. Recent studies on HEAT, TPR, Ankyrin, and Armadillo have contributed to the understanding of the folding of helical repeat proteins. Moreover, constructs composed of simplified “consensus” sequence representations of helical repeat proteins have been found to be well-behaved and highly stable, and facilitate energy dissection using simple nearest-neighbor models. In contrast, little progress has been made towards understanding the folding of β-sheet containing repeat. To determine the folding stability and cooperativity of these proteins, and to better understand the sequence determinants of structure and stability within these ubiquitous families, we have initiated studies on a series of LRR proteins of both naturally occurring and consensus-designed sequences. We find the LRR proteins PP32 and LC1 to be well-behaved, and to fold in a highly cooperative transition that is consistent with a two-state mechanism. NMR H/2H exchange shows the repeating β-strands on the concave surface of both proteins to be more protected than the rest of the molecules, and can be regarded as an exchange-resistant core, whereas the terminal caps and convex structural elements are more labile. However, truncations and sequence substitution demonstrate that the caps significantly influence stability and kinetics. To further simplify our analysis of β-sheet containing repeat protein folding, we designed consensus LRR sequences. On their own, these constructs are unfolded and/or aggregated. By fusing these consensus sequences with naturally occurring LRR protein YopM, we have obtained solublized, folded arrays that exhibit increased stability and drastically decreased unfolding and refolding rates with repeat number. Further studies are needed to dissect the complex folding pathways taken by these constructs.