Structure of a partially unfolded form of <scp><i>E</i></scp><i>scherichia coli</i> dihydrofolate reductase provides insight into its folding pathway

Kasper, Joseph R.; Liu, Pei-Fen; Park, Chiwook

doi:10.1002/pro.2555

Cited by 11 publications

(45 citation statements)

References 42 publications

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“…Proteolysis of CP18 and CP87 occurs without accumulating any detectable intermediates (Fig. S3 Supporting Information) as we previously observed with wild‐type DHFR . Lack of detectable intermediates suggests that the initial cleavage is much slower than the subsequent cleavages.…”

Section: Resultssupporting

confidence: 76%

“…For simplicity, we refer to the cysteine‐free DHFR as wild‐type in this study. Our previous study on native‐state partial unfolding of DHFR was also carried out with the cysteine‐free DHFR . We generated the expression constructs for the three circular permutants (CP18, CP37, and CP87) using a polymerase chain reaction (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…When the cleavable form is a partially unfolded form, Δ G C‐N ° is less than the global stability of the protein (Δ G U‐N °), and m C‐N is also less than the m ‐value for global unfolding ( m U‐N ). We have successfully investigated the energetics of partial unfolding of DHFR using native‐state proteolysis in our previous studies …”

Section: Resultsmentioning

confidence: 99%

“…The positions of the new termini were selected based on the structures of the native and the partially unfolded forms of DHFR. We have previously determined the structure of a partially unfolded form of DHFR using native‐state proteolysis . The native‐state partial unfolding of DHFR involves unfolding of the Met20 (residue 10–24) and F‐G loops (residues 117–131) (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…The native‐state partial unfolding of DHFR involves unfolding of the Met20 (residue 10–24) and F‐G loops (residues 117–131) (Fig. ) . Also, the Met20 loop is dynamic even in the native form .…”

Section: Introductionmentioning

confidence: 99%

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Effect of circular permutations on transient partial unfolding in proteins

et al. 2016

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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 threedimensional 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.

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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Effect of circular permutations on transient partial unfolding in proteins

et al. 2016

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Thermal Adaptation of Enzymes: Impacts of Conformational Shifts on Catalytic Activation Energy and Optimum Temperature

Maffucci

Laage

Sterpone

et al. 2020

Chemistry A European J

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Thermal adaptation of enzymes is essential for both living organism development in extreme conditions and efficient biocatalytic applications. However,t he molecular mechanisms leading to as hift in catalytic activity optimum temperatures remain unclear,a nd there is increasing experimental evidence that thermala daptation involves complex changes in both structural and reactive properties. Here, ac ombination of enhanced protein conformational sampling with an explicit chemical reaction description was applied to mesophilic and thermophilic homologues of the dihydrofolater eductase enzyme, and aq uantitative description of the stability and catalytic activity shifts between ho-mologues was obtained. The key role played by temperature-induceds hifts in protein conformational distributions is revealed. In contrast with pictures focusing on protein flexibility and dynamics, it is shown that while the homologues' reactionf ree energies are similar, the striking discrepancy between their activation energies is caused by their different conformational changes with temperature. An analytic model is proposedthat combines catalytic activity and structural stability,a nd which quantitativelyp redicts the shift in homologue optimum temperatures.I ti ss hown that this generalm odel provides am olecular explanation of changes in optimum temperatures for several other enzymes.

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Ligand binding to a high‐energy partially unfolded protein

Kasper

Park

2014

Protein Science

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The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP 1 as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP 1 and found that NADP 1 binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP 1 is diminished upon partial unfolding. Based on known crystallographic structures of NADP 1 -bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine-binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP 1 . Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high-energy non-native forms.

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

Cited by 11 publications

References 42 publications

Effect of circular permutations on transient partial unfolding in proteins

Effect of circular permutations on transient partial unfolding in proteins

Thermal Adaptation of Enzymes: Impacts of Conformational Shifts on Catalytic Activation Energy and Optimum Temperature

Ligand binding to a high‐energy partially unfolded protein

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