Extreme Temperature Tolerance of a Hyperthermophilic Protein Coupled to Residual Structure in the Unfolded State

Wallgren, Marcus; Ådén, Jörgen; Pylypenko, Olena; Mikaelsson, Therese; Johansson, Lennart B.‐Å.; Rak, Alexey; Wolf‐Watz, Magnus

doi:10.1016/j.jmb.2008.04.007

Cited by 19 publications

(35 citation statements)

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“…1): for W74C, where Cys74 was labeled with BODIPY, the Trp58-Cys74 distance was probed, and for F10C/W74F, Cys10 was labeled with BODIPY and the Trp58-Cys10 distance was measured. It was demonstrated in earlier work that these mutations and their labeling do not disturb the folded structure of the protein (20). In that study, moreover, these S16 variants were shown to unfold in apparent two-state equilibrium reactions (20).…”

Section: Resultsmentioning

confidence: 64%

“…It was demonstrated in earlier work that these mutations and their labeling do not disturb the folded structure of the protein (20). In that study, moreover, these S16 variants were shown to unfold in apparent two-state equilibrium reactions (20).…”

Section: Resultsmentioning

confidence: 64%

“…The S16 mutants were prepared as previously described (20). The buffer contained 30 mM NaAc and 50 mM NaCl at pH 5.5, with or without addition of 200 mg/ml dextran 20 and with varying urea concentrations.…”

Section: Sample Preparationmentioning

confidence: 99%

“…This indicates that the variants are stabilized in the presence of crowding agent (DG and midpoint values are summarized in Table 1). It is important to note that in the presence of crowding FIGURE 1 Intramolecular distances displayed on the crystal structure of wild-type S16 (20). The side chain of Trp58 (FRET donor) is colored in green.…”

Section: Equilibrium Unfoldingmentioning

confidence: 99%

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Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding

Mikaelsson

Ådén

Johansson

et al. 2013

Biophysical Journal

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Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.

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Section: Resultsmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 64%

Section: Sample Preparationmentioning

confidence: 99%

Section: Equilibrium Unfoldingmentioning

confidence: 99%

See 2 more Smart Citations

Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding

Mikaelsson

Ådén

Johansson

et al. 2013

Biophysical Journal

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“…The plasmid was kindly provided by A. C. Rosenzweig (Northwestern University, Evanston, IL). Aquifex aeolicus S16 was purified following established procedures (39), and horse heart cytochrome c was purchased from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

Palm¹,

Weise²,

Lundin

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Cisplatin (cisPt), PtðNH 3 Þ 2 Cl 2 , is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1's metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.

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Fundamentals of Protein Folding

Uversky¹

2014

Protein Aggregation in Bacteria

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Extreme Temperature Tolerance of a Hyperthermophilic Protein Coupled to Residual Structure in the Unfolded State

Cited by 19 publications

References 50 publications

Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding

Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding

Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

Fundamentals of Protein Folding

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