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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

The three-state transition of DmChd64 and TcChd64 at different GdmCl concentrations.Populations of native, unfolded and intermediate molecules for DmChd64 (A) and TcChd64 (B) were expressed as a fraction of all DmChd64 and TcChd64 molecules. The fraction of native (fN) molecules (black squares) was calculated from the fluorescence measurements, using Eq 4, and the fraction of unfolded (fU) molecules (red dots) was calculated from SEC and far-UV CD analyses, using Eq 5. In our case, fN + fU < 1 and the intermediate state fraction (fi) (blue triangles) was defined by Eq 6.
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pone.0137074.g006: The three-state transition of DmChd64 and TcChd64 at different GdmCl concentrations.Populations of native, unfolded and intermediate molecules for DmChd64 (A) and TcChd64 (B) were expressed as a fraction of all DmChd64 and TcChd64 molecules. The fraction of native (fN) molecules (black squares) was calculated from the fluorescence measurements, using Eq 4, and the fraction of unfolded (fU) molecules (red dots) was calculated from SEC and far-UV CD analyses, using Eq 5. In our case, fN + fU < 1 and the intermediate state fraction (fi) (blue triangles) was defined by Eq 6.

Mentions: DmChd64 and TcChd64 unfolding, as measured using a range of techniques, is reversible and complex. Molecules in certain conditions can exist in a well-defined intermediate conformation. Far-UV CD and fluorescence spectroscopy and SEC data were used to calculate populations of molecules under particular states. The percentage of molecules after conformational transition (%D) was plotted against increasing GdmCl concentrations (Fig 5A and 5B), and the transition Cm value for each experiment was calculated (Table 1). The unfolding curves did not overlap. For both DmChd64 and TcChd64, significant differences were found between the fluorescence spectroscopy results and the far-UV CD spectroscopy and SEC results. The fluorescence measurements, which provide insight into protein integrity, revealed that the transition occurred from 0.8–2.0 M GdmCl for DmChd64 and 0.5–2.5 M for TcChd64, with a transition Cm of 1.32 ± 0.03 M and 1.33 ± 0.04 M GdmCl for DmChd64 and TcChd64, respectively (Fig 5A and 5B). The far-UV CD results revealed changes in the secondary structural content. The transition occurred at higher GdmCl concentrations compared with those found by fluorescence. For DmChd64, the transition occurred at 1.4–2.2 M GdmCl, with a Cm of 1.94 ± 0.10 M GdmCl; for TcChd64, the transition occurred at 2.0–3.0 M, with a Cm of 2.57 ± 0.12 M GdmCl (Fig 5A and 5B). Additionally, the transition occurred at higher denaturant concentrations when monitored by SEC, falling within a range of 1.0–3.0 M and 1.5–3.5 M GdmCl, with a Cm of 2.09 ± 0.08 M and 2.57 ± 0.10 M GdmCl, for DmChd64 and TcChd64, respectively. These values indicate that unfolding processes begin with destabilisation of the molecular integrity, followed by the unwinding of secondary structures. Between these two phases, Chd64 exists as an intermediate state population (Fig 6A and 6B) that does not exceed 1.7 M GdmCl for DmChd64 and 1.9 M GdmCl for TcChd64. The intermediate state content is significant and can comprise 75% of the entire population of molecules. Consequently, GdmCl-induced Chd64 unfolding can be defined by a three-state model that includes native, intermediate and unfolded states. In the intermediate state, which resembles a MG conformation, helices formed in the globular core are largely preserved but are not fixed in 3D space [22,23].


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)

The three-state transition of DmChd64 and TcChd64 at different GdmCl concentrations.Populations of native, unfolded and intermediate molecules for DmChd64 (A) and TcChd64 (B) were expressed as a fraction of all DmChd64 and TcChd64 molecules. The fraction of native (fN) molecules (black squares) was calculated from the fluorescence measurements, using Eq 4, and the fraction of unfolded (fU) molecules (red dots) was calculated from SEC and far-UV CD analyses, using Eq 5. In our case, fN + fU < 1 and the intermediate state fraction (fi) (blue triangles) was defined by Eq 6.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0137074.g006: The three-state transition of DmChd64 and TcChd64 at different GdmCl concentrations.Populations of native, unfolded and intermediate molecules for DmChd64 (A) and TcChd64 (B) were expressed as a fraction of all DmChd64 and TcChd64 molecules. The fraction of native (fN) molecules (black squares) was calculated from the fluorescence measurements, using Eq 4, and the fraction of unfolded (fU) molecules (red dots) was calculated from SEC and far-UV CD analyses, using Eq 5. In our case, fN + fU < 1 and the intermediate state fraction (fi) (blue triangles) was defined by Eq 6.
Mentions: DmChd64 and TcChd64 unfolding, as measured using a range of techniques, is reversible and complex. Molecules in certain conditions can exist in a well-defined intermediate conformation. Far-UV CD and fluorescence spectroscopy and SEC data were used to calculate populations of molecules under particular states. The percentage of molecules after conformational transition (%D) was plotted against increasing GdmCl concentrations (Fig 5A and 5B), and the transition Cm value for each experiment was calculated (Table 1). The unfolding curves did not overlap. For both DmChd64 and TcChd64, significant differences were found between the fluorescence spectroscopy results and the far-UV CD spectroscopy and SEC results. The fluorescence measurements, which provide insight into protein integrity, revealed that the transition occurred from 0.8–2.0 M GdmCl for DmChd64 and 0.5–2.5 M for TcChd64, with a transition Cm of 1.32 ± 0.03 M and 1.33 ± 0.04 M GdmCl for DmChd64 and TcChd64, respectively (Fig 5A and 5B). The far-UV CD results revealed changes in the secondary structural content. The transition occurred at higher GdmCl concentrations compared with those found by fluorescence. For DmChd64, the transition occurred at 1.4–2.2 M GdmCl, with a Cm of 1.94 ± 0.10 M GdmCl; for TcChd64, the transition occurred at 2.0–3.0 M, with a Cm of 2.57 ± 0.12 M GdmCl (Fig 5A and 5B). Additionally, the transition occurred at higher denaturant concentrations when monitored by SEC, falling within a range of 1.0–3.0 M and 1.5–3.5 M GdmCl, with a Cm of 2.09 ± 0.08 M and 2.57 ± 0.10 M GdmCl, for DmChd64 and TcChd64, respectively. These values indicate that unfolding processes begin with destabilisation of the molecular integrity, followed by the unwinding of secondary structures. Between these two phases, Chd64 exists as an intermediate state population (Fig 6A and 6B) that does not exceed 1.7 M GdmCl for DmChd64 and 1.9 M GdmCl for TcChd64. The intermediate state content is significant and can comprise 75% of the entire population of molecules. Consequently, GdmCl-induced Chd64 unfolding can be defined by a three-state model that includes native, intermediate and unfolded states. In the intermediate state, which resembles a MG conformation, helices formed in the globular core are largely preserved but are not fixed in 3D space [22,23].

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