Product Inhibition in Native-State Proteolysis

Kasper, Joseph R.; Andrews, Elizabeth C.; Park, Chiwook

doi:10.1371/journal.pone.0111416

Cited by 9 publications

(8 citation statements)

References 33 publications

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“…The apparent rate constants of proteolysis ( k obs ) were determined by fitting the change in band intensity over time to a first‐order rate equation in OriginPro 8.5.1 (OriginLab; Northampton, MA). We have recently reported that, due to product inhibition, proteolysis of intact proteins may deviate from first‐order kinetics, and k obs may be underestimated . In DHFR, however, we have shown that Δ G op ° is still well consistent with Δ G unf °, which confirms that Δ G op ° is determined reliably by native‐state proteolysis.…”

Section: Methodssupporting

confidence: 69%

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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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Section: Methodssupporting

confidence: 69%

“…We have recently reported that, due to product inhibition, proteolysis of intact proteins may deviate from first-order kinetics, and k obs may be underestimated. 38 In DHFR, however, we have shown that DG op is still well consistent with DG unf , 16 which confirms that DG op is determined reliably by native-state proteolysis.…”

Section: Proteolysissupporting

confidence: 71%

Ligand binding to a high‐energy partially unfolded protein

Kasper

Park

2014

Protein Science

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“…The addition of both charges to K86Q/D108N RNase H * decreases the proteolysis rate by less than twofold, which correspond to increase in Δ G C‐N ° by ∼0.3 kcal/mol. It is noteworthy that Δ G C‐N ° values from native‐state proteolysis have some uncertainty due to the choice of k int and the effect of product inhibition on determination of k p . However, the uncertainty in Δ G C‐N ° does not affect ΔΔ G C‐N ° values because the systematic errors in Δ G C‐N ° determination are cancelled out in calculation of ΔΔ G C‐N °…”

Section: Resultsmentioning

confidence: 99%

“…It is noteworthy that DG C-N 8 values from native-state proteolysis have some uncertainty due to the choice of k int and the effect of product inhibition on determination of k p . 39 However, the uncertainty in DG C-N 8 does not affect DDG C-N 8 values because the systematic errors in DG C-N 8 determination are cancelled out in calculation of DDG C-N 8. 28 The energy diagram of the free energy of the native and cleavable forms relative to that of the unfolded form shows the effect of the salt bridge and the salt concentration on each form (Fig.…”

Section: Contribution Of the Lys86-asp108 Salt Bridge To Global Stabimentioning

confidence: 99%

Salt bridge as a gatekeeper against partial unfolding

2016

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Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native-state partial unfolding in a cysteine-free version of Escherichia coli ribonuclease H (RNase H*). Thermolysin cleaves a protruding loop of RNase H* through transient partial unfolding under native conditions. Lys86 and Asp108 in RNase H* form a partially buried salt bridge that tethers the protruding loop. Investigation of the global stability of K86Q/D108N RNase H* showed that the salt bridge does not significantly contribute to the global stability. However, K86Q/D108N RNase H* is greatly more susceptible to proteolysis by thermolysin than wild-type RNase H* is. The free energy for partial unfolding determined by native-state proteolysis indicates that the salt bridge significantly increases the energy for partial unfolding by destabilizing the partially unfolded form. Double mutant cycles with single and double mutations of the salt bridge suggest that the partially unfolded form is destabilized due to a significant decrease in the interaction energy between Lys86 and Asp108 upon partial unfolding. This study demonstrates that, even in the case that a salt bridge does not contribute to the global stability, the salt bridge may function as a gatekeeper against partial unfolding that disturbs the optimal geometry of the salt bridge.

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“…Peptidase R yielded the highest free amino acids compared to other exo-proteases tested. Alcalase 2.4L FG was also selected due to reported specificity towards hydrophobic amino acids (Kasper et al 2014 ).…”

Section: Methodsmentioning

confidence: 99%

Production of hydrophobic amino acids from biobased resources: wheat gluten and rubber seed proteins

WIDYARANI

Sari

Ratnaningsih

et al. 2016

Appl Microbiol Biotechnol

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Protein hydrolysis enables production of peptides and free amino acids that are suitable for usage in food and feed or can be used as precursors for bulk chemicals. Several essential amino acids for food and feed have hydrophobic side chains; this property may also be exploited for subsequent separation. Here, we present methods for selective production of hydrophobic amino acids from proteins. Selectivity can be achieved by selection of starting material, selection of hydrolysis conditions, and separation of achieved hydrolysate. Several protease combinations were applied for hydrolysis of rubber seed protein concentrate, wheat gluten, and bovine serum albumin (BSA). High degree of hydrolysis (>50 %) could be achieved. Hydrophobic selectivity was influenced by the combination of proteases and by the extent of hydrolysis. Combination of Pronase and Peptidase R showed the highest selectivity towards hydrophobic amino acids, roughly doubling the content of hydrophobic amino acids in the products compared to the original substrates. Hydrophobic selectivity of 0.6 mol-hydrophobic/mol-total free amino acids was observed after 6 h hydrolysis of wheat gluten and 24 h hydrolysis of rubber seed proteins and BSA. The results of experiments with rubber seed proteins and wheat gluten suggest that this process can be applied to agro-industrial residues.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7441-8) contains supplementary material, which is available to authorized users.

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Product Inhibition in Native-State Proteolysis

Cited by 9 publications

References 33 publications

Ligand binding to a high‐energy partially unfolded protein

Ligand binding to a high‐energy partially unfolded protein

Salt bridge as a gatekeeper against partial unfolding

Production of hydrophobic amino acids from biobased resources: wheat gluten and rubber seed proteins

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