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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.

SUBMITTER: Chen C

PROVIDER: S-EPMC4972204 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Effect of circular permutations on transient partial unfolding in proteins.

Chen Chen C Yun Jung-Hun JH Kim Jae-Hoon JH Park Chiwook C

Protein science : a publication of the Protein Society 20160524 8

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 perm ...[more]

PMID: 27164316

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Project description:Circular permutation (CP) refers to situations in which the termini of a protein are relocated to other positions in the structure. CP occurs naturally and has been artificially created to study protein function, stability and folding. Recently CP is increasingly applied to engineer enzyme structure and function, and to create bifunctional fusion proteins unachievable by tandem fusion. CP is a complicated and expensive technique. An intrinsic difficulty in its application lies in the fact that not every position in a protein is amenable for creating a viable permutant. To examine the preferences of CP and develop CP viability prediction methods, we carried out comprehensive analyses of the sequence, structural, and dynamical properties of known CP sites using a variety of statistics and simulation methods, such as the bootstrap aggregating, permutation test and molecular dynamics simulations. CP particularly favors Gly, Pro, Asp and Asn. Positions preferred by CP lie within coils, loops, turns, and at residues that are exposed to solvent, weakly hydrogen-bonded, environmentally unpacked, or flexible. Disfavored positions include Cys, bulky hydrophobic residues, and residues located within helices or near the protein's core. These results fostered the development of an effective viable CP site prediction system, which combined four machine learning methods, e.g., artificial neural networks, the support vector machine, a random forest, and a hierarchical feature integration procedure developed in this work. As assessed by using the hydrofolate reductase dataset as the independent evaluation dataset, this prediction system achieved an AUC of 0.9. Large-scale predictions have been performed for nine thousand representative protein structures; several new potential applications of CP were thus identified. Many unreported preferences of CP are revealed in this study. The developed system is the best CP viability prediction method currently available. This work will facilitate the application of CP in research and biotechnology.

Dataset Information

Effect of circular permutations on transient partial unfolding in proteins.

Publications

Effect of circular permutations on transient partial unfolding in proteins.

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