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Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway.

ABSTRACT: Proteins frequently fold via folding intermediates that correspond to local minima on the conformational energy landscape. Probing the structure of the partially unfolded forms in equilibrium under native conditions can provide insight into the properties of folding intermediates. To elucidate the structures of folding intermediates of Escherichia coli dihydrofolate reductase (DHFR), we investigated transient partial unfolding of DHFR under native conditions. We probed the structure of a high-energy conformation susceptible to proteolysis (cleavable form) using native-state proteolysis. The free energy for unfolding to the cleavable form is clearly less than that for global unfolding. The dependence of the free energy on urea concentration (m-value) also confirmed that the cleavable form is a partially unfolded form. By assessing the effect of mutations on the stability of the partially unfolded form, we found that native contacts in a hydrophobic cluster formed by the F-G and Met-20 loops on one face of the central ?-sheet are mostly lost in the partially unfolded form. Also, the folded region of the partially unfolded form is likely to have some degree of structural heterogeneity. The structure of the partially unfolded form is fully consistent with spectroscopic properties of the near-native kinetic intermediate observed in previous folding studies of DHFR. The findings suggest that the last step of the folding of DHFR involves organization in the structure of two large loops, the F-G and Met-20 loops, which is coupled with compaction of the rest of the protein.

SUBMITTER: Kasper JR

PROVIDER: S-EPMC4253813 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway.

Kasper Joseph R JR Liu Pei-Fen PF Park Chiwook C

Protein science : a publication of the Protein Society 20141018 12

Proteins frequently fold via folding intermediates that correspond to local minima on the conformational energy landscape. Probing the structure of the partially unfolded forms in equilibrium under native conditions can provide insight into the properties of folding intermediates. To elucidate the structures of folding intermediates of Escherichia coli dihydrofolate reductase (DHFR), we investigated transient partial unfolding of DHFR under native conditions. We probed the structure of a high-en ...[more]

PMID: 25252157

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Project description:Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecDHFR.

Dataset Information

Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway.

Publications

Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway.

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