Small globular proteins have many contacts between residues that are distant in primary structure. These contacts create a complex network among sequence-distant segments of secondary structure and are expected to promote cooperative folding. Although repeat proteins, which are made up of tandem modular units, lack sequence-distant contacts, several repeat proteins of considerable length have been shown to undergo cooperative two-state folding.
To explore the limits of cooperativity in repeat proteins, we have studied a large LRR protein, YopM, a bacterial virulence factor secreted by Yersinia pestis. YopM contains 15 tandem LRR units each composed of 20 or 22 residues. YopM has a molecular architecture that resembles a horseshoe with a slight helical twist. The individual LRR units have β-strands that stack to form a continuous parallel sheet along the concave surface. This architecture provides a system in which energetic contributions of small structural segments can be determined.
Despite its linear architecture and large size, we show that wild-type YopM folds by an equilibrium two-state mechanism by monitoring both chemically and thermally-induced equilibrium unfolding transitions, along with differential scanning calorimetry. Stopped-flow kinetics monitored by fluorescence shows that YopM folds by a simple equilibrium mechanism, the folding kinetic mechanism is more complex, revealing at least one transient intermediate. To investigate how electrostatics influence stability in a changed but elongated protein, the origin of an unusually large salt dependence of the free energy of folding was determined. Rather than site-specific binding or solvent effects, electrostatic interactions account for the large stabilization by salt.
C-terminal deletion constructs were studied to gain insight into the physical origins of protein folding cooperativity and the distribution of stability over the LRRs of YopM. Surprisingly, the stability was found to be highly non-uniform in distribution. Despite this heterogeneous distribution, cooperativity is maintained by key internal and terminal capping motifs.
To probe the distribution of interaction energy between the interfaces of β-strand containing repeat proteins, we studied internal deletions of three LRRs from YopM. Contrary to our expectation that deletion of structured repeats should be destabilizing, we found both an example of a neutral internal deletion and one which is stabilizing. Our results demonstrate that not all LRR interfaces are equivalent, which has implications for protein evolution by repeat rearrangement and protein design.
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 70/10, Dissertation Abstracts International|
|Keywords:||Cooperativity, Heterogeneous stability, Leucine-rich repeat, Protein folding|
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