title: Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability.
abstract: The leucine-rich repeat domain of PP32 is composed of five β-strand-containing repeats anchored by terminal caps. These repeats differ in sequence but are similar in structure, providing a means to connect topology, sequence, and folding pathway selection. Through kinetic studies of PP32, we find folding to be rate-limited by the formation of an on-pathway intermediate. Destabilizing core substitutions reveal a transition state ensemble that is highly polarized toward the C-terminal repeat and cap. To determine if this nucleus for folding corresponds to the most stable region of PP32, we monitored amide hydrogen exchange by NMR spectroscopy. Indeed, we find the highest protection to be biased toward the C terminus. Sequence manipulations that destabilize the C terminus spread out the transition state toward the middle of the protein. Consistent with results for helical ankyrin repeat proteins, these results suggest that local stabilities determine folding pathways.
authors.affiliation: Departments of Biology and Chemistry, Syracuse University, Syracuse, NY 13244; and.
authors.forename: Thuy Phuong
authors.initials: TP
authors.lastname: Dao
authors.affiliation: The Johns Hopkins University Biomolecular NMR Center and.
authors.forename: Ananya
authors.initials: A
authors.lastname: Majumdar
authors.affiliation: Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218 barrick@jhu.edu.
authors.forename: Doug
authors.initials: D
authors.lastname: Barrick
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
chemical_list: C087425:ANP32A protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D006859:Hydrogen
keywords: LRR proteins; PP32; folding pathway selection; on-pathway intermediate; polarized transition state