C-terminal deletion of leucine-rich repeats from YopM reveals a heterogeneous distribution of stability in a cooperatively folded protein.
Terminal deletions of units from alpha-helical repeat proteins have provided insight into the physical origins of their cooperativity. To test if the same principles governing cooperativity apply to beta-sheet-containing repeat proteins, we have created a series of C-terminal deletion constructs from a large leucine-rich repeat (LRR) protein, YopM. We have examined the structure and stability of the resulting deletion constructs by a combination of solution spectroscopy, equilibrium denaturation studies, and limited proteolysis. Surprisingly, a high degree of nonuniformity was found in the stability distribution of YopM. Unlike previously studied repeat proteins, we identified several key LRR that on deletion disrupt nearby structure, at distances as far away as up to three repeats, in YopM. This partial unfolding model is supported by limited proteolysis studies and by point substitution in repeats predicted to be disordered as a result of deletion of adjacent repeats. We show that key internal- and terminal-caps must be present to maintain the structural integrity in adjacent regions (roughly four LRRs long) of decreased stability. The finding that full-length YopM maintains a high level of cooperativity in equilibrium unfolding underscores the importance of interfacial interactions in stabilizing locally unstable regions of structure.
Kloss
Ellen
E
T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Barrick
Doug
D
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D000090263:Leucine-Rich Repeat Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D015007:Yersinia / Q000737:chemistry*
D001425:Bacterial Outer Membrane Proteins; D000090263:Leucine-Rich Repeat Proteins; D011506:Proteins; C060772:yopM protein, Yersinia