title: Effect of circular permutations on transient partial unfolding in proteins.
abstract: Under native conditions, proteins can undergo transient partial unfolding, which may cause proteins to misfold or aggregate. A change in sequence connectivity by circular permutation may affect the energetics of transient partial unfolding in proteins without altering the three-dimensional structures. Using Escherichia coli dihydrofolate reductase (DHFR) as a model system, we investigated how circular permutation affects transient partial unfolding in proteins. We constructed three circular permutants, CP18, CP37, and CP87, with the new N-termini at residue 18, 37, and 87, respectively, and probed transient partial unfolding by native-state proteolysis. The new termini in CP18, CP37, and CP87 are within, near, and distal to the Met20 loop, which is known to be dynamic and also part of the region that undergoes transient unfolding in wild-type DHFR. The stabilities of both native and partially unfolded forms of CP18 are similar to those of wild-type DHFR, suggesting that the influence of introducing new termini in a dynamic region to the protein is minimal. CP37 has a significantly more accessible partially unfolded form than wild-type DHFR, demonstrating that introducing new termini near a dynamic region may promote transient partial unfolding. CP87 has significantly destabilized native and partially unfolded forms, confirming that modification of the folded region in a partially unfolded form destabilizes the partially unfolded form similar to the native form. Our findings provide valuable guidelines to control transient partial unfolding in designing circular permutants in proteins.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, 47907.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: Faculty of Biotechnology, College of Applied Life Science, SARI, Jeju National University, Jeju-Do, 690-756, Korea.
authors.forename: Jung-Hun
authors.initials: JH
authors.lastname: Yun
authors.affiliation: Faculty of Biotechnology, College of Applied Life Science, SARI, Jeju National University, Jeju-Do, 690-756, Korea.
authors.forename: Jae-Hoon
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, 47907.
authors.forename: Chiwook
authors.initials: C
authors.lastname: Park
mesh_terms: D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D000072417:Protein Domains; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D059748:Proteolysis; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase
keywords: DHFR; circular permutation; native-state proteolysis; partial unfolding; protein engineering; proteolytic susceptibility