ALPHA-HELICAL REPEAT PROTEINFOLDING AND TURNOVER:A THERMODYNAMIC ANALYSIS OFNATURAL AND UNNATURAL REPEAT ARCHITECTURES

[摘要] Studies using repeat proteins have been invaluable to our understanding of protein folding thermodynamics.Here we present work investigating the origins behind protein folding stabilities and protein turnover for designed alpha-helical repeat proteins.We have also developed a novel protein purification method exploiting polyaspartate and polyglutamate affinity tags binding on hydroxyapatite. Consensus ankyrin repeat proteins are designed repeats with a naturally occurring structural conformation and remarkably high stability.We observe a direct relationship between levels of sequence conservation with changes in two-state folding stability revealed by circular dichroism (CD) spectroscopy with single-substituted repeats.In addition, one-dimensional Ising analysis revealed that consensus amino acids preferentially stabilize the repeat interfaces more than the intrinsic repeats.Consistent with previous NMR findings, we observed high interfacial stabilities conferred from residues involved in a buried polar network.Together, these findings support the importance of conserved buried interfacial polar residues in natural repeat protein conformations. In contrast to natural repeat structures, applying Ising analysis to CD data for repeat proteins with unnatural conformations revealed stabilizing intrinsic repeat stabilities.The fast folding kinetics observed for these repeat proteins support a barrierless folding pathway.These data uncover short-range interactions as the origins of extreme stability for these unnatural repeat architectures. We investigated the relationship stability has with protein turnover using an in vitro assay to degrade consensus ankyrin repeats with E. coli Lon protease.While we observed an expected increase in rates of degradation with a decrease in global stability, however the distance-dependence of stability on proteolysis remains an open question. We have developed a novel purification method exploiting polyaspartate and polyglutamate binding to hydroxyapatite.We observed binding independent of terminal placement, an increased affinity with longer tag length, and positive detection of polyglutamate tags by Western blot.Because the fusion-tag decreased rates of E. coli adenylate kinase catalysis, we recommend tag removal from active enzymes. Overall, these results provide insight into the relationship between protein sequence and stability for a variety of protein architectures.It is our hope that the findings and molecular tools developed here will prove useful for future protein engineering studies.

[发布机构] Johns Hopkins University

[效力级别] repeat protein

[关键词] protein folding;repeat protein;alpha-helical;circular dichroism;ising analysis;SDS-PAGE;hydroxyapatite;polyglutamate;polyaspartate;Lon protease;degron;proteolysis;protein affinity purification tag;Biology