期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：18
页码：E2298-E2306
DOI：10.1073/pnas.1412165112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceProteins fold to their native structure in significantly shorter timescales than expected for a random search mechanism. Rapid folding, which is likely to be important to optimize yield and avoid misfolded, aggregated states, has been proposed to result from specific folding pathways. However, mapping such pathways and understanding the principles by which they are selected remain central problems in protein science. Repeat proteins, containing tandem repeated structural units, greatly simplify folding studies. Here, we exploit the linear architecture of a {beta}-strand-containing leucine-rich repeat protein and discover that folding is highly polarized toward the C terminus. By measuring the energy distribution, we observe that polarization is guided by local stability. The leucine-rich repeat domain of PP32 is composed of five {beta}-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.
