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Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails.

Tarczewska A, Kozłowska M, Dobryszycki P, Kaus-Drobek M, Dadlez M, Ożyhar A - PLoS ONE (2015)

Bottom Line: Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways.Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations.The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wrocław, Poland.

ABSTRACT
Two major lipophilic hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64) and Tribolium castaneum (TcChd64). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH) domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV) circular dichroism (CD) spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC) revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG)-like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners.

No MeSH data available.


Related in: MedlinePlus

Changes in the hydrodynamic properties of DmChd64 and TcChd64 induced by GdmCl.(A), (C) SEC analysis of DmChd64 (A) and TcChd64 (B) in the presence of GdmCl. The curves represent profiles of DmChd64 and TcChd64 chromatographed at different GdmCl concentrations. The blue lines represent elution peaks of renatured proteins chromatographed in buffer A. Purified DmChd64 and TcChd64 (0.5 mg/ml in buffer A with an appropriate GdmCl concentration) were injected into a Superdex 200 10/300 GL column equilibrated with buffer A of the same denaturant concentration for each sample at room temperature, with a flow rate of 0.5 ml/min. (B), (D) Using the calibration curve (see Materials and Methods) and the elution volumes determined in (A) and (C), the Rs values of DmCh64 (B) and TcChd64 (D) were calculated and plotted against the GdmCl concentration.
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pone.0137074.g004: Changes in the hydrodynamic properties of DmChd64 and TcChd64 induced by GdmCl.(A), (C) SEC analysis of DmChd64 (A) and TcChd64 (B) in the presence of GdmCl. The curves represent profiles of DmChd64 and TcChd64 chromatographed at different GdmCl concentrations. The blue lines represent elution peaks of renatured proteins chromatographed in buffer A. Purified DmChd64 and TcChd64 (0.5 mg/ml in buffer A with an appropriate GdmCl concentration) were injected into a Superdex 200 10/300 GL column equilibrated with buffer A of the same denaturant concentration for each sample at room temperature, with a flow rate of 0.5 ml/min. (B), (D) Using the calibration curve (see Materials and Methods) and the elution volumes determined in (A) and (C), the Rs values of DmCh64 (B) and TcChd64 (D) were calculated and plotted against the GdmCl concentration.

Mentions: We used SEC to investigate the structural changes that occur during the native-to-unfolded transition of the protein molecules. Using SEC, molecules with different levels of compactness are separated [21]. Previously, we found that the Rs values of DmChd64 and TcChd64 are larger than the theoretical values [8]. Consequently, the molecular volumes are also larger compared with globular equivalents of the same theoretical mass. We correlated this with the existence of a putative partially disordered conformation demonstrated by HDX. Increasing GdmCl concentrations strongly affected the Rs of Chd64. Notably, at 1.25 M and 1.50 M GdmCl for DmChd64 and TcChd64, respectively, the elution peaks become broader and asymmetrical (Fig 4A and 4C) indicating that both Chd64 proteins undergo significant conformational changes and that Chd64 protein populations become heterogeneous during the native-to-unfolded transition. Some additional states exist that are neither native nor unfolded (Fig 4A, 1.25–2 M GdmCl and 4B, 1.5–2 M GdmCl). The appearance of such discrete conformational states suggests that some conditions alter the shapes of molecules, indicating that Chd64 is a relatively flexible, pliable molecule that can exist in different conformational states. In a buffer containing 3.0–6.0 M GdmCl, the hydrodynamic volume of DmChd64 significantly increased and peaks became symmetrical again, suggesting that the equilibrium between the native and unfolded forms shifted towards the latter from (Fig 4A). A similar pattern was observed for TcChd64 (Fig 4C), although the respective transition occurred at higher GdmCl concentrations. At 1.25 M GdmCl, at which point DmChd64 was undergoing conformational changes and existed in different conformational forms (Fig 4A), TcChd64 still generated a symmetrical peak (Fig 4C). However, different conformers of TcChd64 were present at 1.5 M GdmCl. At 3.0 M GdmCl, the elution volume decreased, indicating that TcChd64 was already denatured. The apparent Rs increased twice during the transition between the native and denatured forms, reaching 22.76 ± 0.68 Å for native DmChd64 to 43.83 ± 0.90 Å for denatured proteins at 6.0 M GdmCl. The Rs of TcChd64 increased from 23.75 ± 0.71 Å for native proteins to 46.87 ± 0.92 Å for proteins in 6 M GdmCl. These GdmCl-induced unfolding study results revealed that the native-to-unfolded transition is a complex process for both Chd64 proteins. Fluorescence spectroscopy results showed that 1.3 M of GdmCl induced destabilisation of the local tryptophan residue environment (Fig 3A and 3B). This, however, does not significantly influence the global architecture of proteins that depends on secondary structural elements. At 1.3 M, these elements were primarily preserved in both Chd64 proteins; however, the population of Chd64 molecules became heterogeneous (Fig 4A and 4C), indicating that both Chd64 proteins could adopt distinct conformational states under certain conditions.


Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails.

Tarczewska A, Kozłowska M, Dobryszycki P, Kaus-Drobek M, Dadlez M, Ożyhar A - PLoS ONE (2015)

Changes in the hydrodynamic properties of DmChd64 and TcChd64 induced by GdmCl.(A), (C) SEC analysis of DmChd64 (A) and TcChd64 (B) in the presence of GdmCl. The curves represent profiles of DmChd64 and TcChd64 chromatographed at different GdmCl concentrations. The blue lines represent elution peaks of renatured proteins chromatographed in buffer A. Purified DmChd64 and TcChd64 (0.5 mg/ml in buffer A with an appropriate GdmCl concentration) were injected into a Superdex 200 10/300 GL column equilibrated with buffer A of the same denaturant concentration for each sample at room temperature, with a flow rate of 0.5 ml/min. (B), (D) Using the calibration curve (see Materials and Methods) and the elution volumes determined in (A) and (C), the Rs values of DmCh64 (B) and TcChd64 (D) were calculated and plotted against the GdmCl concentration.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556635&req=5

pone.0137074.g004: Changes in the hydrodynamic properties of DmChd64 and TcChd64 induced by GdmCl.(A), (C) SEC analysis of DmChd64 (A) and TcChd64 (B) in the presence of GdmCl. The curves represent profiles of DmChd64 and TcChd64 chromatographed at different GdmCl concentrations. The blue lines represent elution peaks of renatured proteins chromatographed in buffer A. Purified DmChd64 and TcChd64 (0.5 mg/ml in buffer A with an appropriate GdmCl concentration) were injected into a Superdex 200 10/300 GL column equilibrated with buffer A of the same denaturant concentration for each sample at room temperature, with a flow rate of 0.5 ml/min. (B), (D) Using the calibration curve (see Materials and Methods) and the elution volumes determined in (A) and (C), the Rs values of DmCh64 (B) and TcChd64 (D) were calculated and plotted against the GdmCl concentration.
Mentions: We used SEC to investigate the structural changes that occur during the native-to-unfolded transition of the protein molecules. Using SEC, molecules with different levels of compactness are separated [21]. Previously, we found that the Rs values of DmChd64 and TcChd64 are larger than the theoretical values [8]. Consequently, the molecular volumes are also larger compared with globular equivalents of the same theoretical mass. We correlated this with the existence of a putative partially disordered conformation demonstrated by HDX. Increasing GdmCl concentrations strongly affected the Rs of Chd64. Notably, at 1.25 M and 1.50 M GdmCl for DmChd64 and TcChd64, respectively, the elution peaks become broader and asymmetrical (Fig 4A and 4C) indicating that both Chd64 proteins undergo significant conformational changes and that Chd64 protein populations become heterogeneous during the native-to-unfolded transition. Some additional states exist that are neither native nor unfolded (Fig 4A, 1.25–2 M GdmCl and 4B, 1.5–2 M GdmCl). The appearance of such discrete conformational states suggests that some conditions alter the shapes of molecules, indicating that Chd64 is a relatively flexible, pliable molecule that can exist in different conformational states. In a buffer containing 3.0–6.0 M GdmCl, the hydrodynamic volume of DmChd64 significantly increased and peaks became symmetrical again, suggesting that the equilibrium between the native and unfolded forms shifted towards the latter from (Fig 4A). A similar pattern was observed for TcChd64 (Fig 4C), although the respective transition occurred at higher GdmCl concentrations. At 1.25 M GdmCl, at which point DmChd64 was undergoing conformational changes and existed in different conformational forms (Fig 4A), TcChd64 still generated a symmetrical peak (Fig 4C). However, different conformers of TcChd64 were present at 1.5 M GdmCl. At 3.0 M GdmCl, the elution volume decreased, indicating that TcChd64 was already denatured. The apparent Rs increased twice during the transition between the native and denatured forms, reaching 22.76 ± 0.68 Å for native DmChd64 to 43.83 ± 0.90 Å for denatured proteins at 6.0 M GdmCl. The Rs of TcChd64 increased from 23.75 ± 0.71 Å for native proteins to 46.87 ± 0.92 Å for proteins in 6 M GdmCl. These GdmCl-induced unfolding study results revealed that the native-to-unfolded transition is a complex process for both Chd64 proteins. Fluorescence spectroscopy results showed that 1.3 M of GdmCl induced destabilisation of the local tryptophan residue environment (Fig 3A and 3B). This, however, does not significantly influence the global architecture of proteins that depends on secondary structural elements. At 1.3 M, these elements were primarily preserved in both Chd64 proteins; however, the population of Chd64 molecules became heterogeneous (Fig 4A and 4C), indicating that both Chd64 proteins could adopt distinct conformational states under certain conditions.

Bottom Line: Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways.Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations.The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wrocław, Poland.

ABSTRACT
Two major lipophilic hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64) and Tribolium castaneum (TcChd64). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH) domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV) circular dichroism (CD) spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC) revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG)-like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners.

No MeSH data available.


Related in: MedlinePlus