Biochemical and Biophysical Characterization of the Caveolin-2 Interaction with Membranes and Analysis of the Protein Structural Alteration by the Presence of Cholesterol
Abstract
:1. Introduction
2. Results
2.1. Purification of Human Recombinant Cav-2
2.2. Structural Predictions Based on the Cav-2 Amino Acid Sequence
2.2.1. Homology Analysis
2.2.2. Prediction of Intrinsically Disordered Segments
2.2.3. Hydropathy Analysis
2.3. Far-UV-Circular Dichroism (CD) Spectra of Cav-2
2.4. Lipids Stabilize the Strucuture Cav-2
2.5. Cav-2 Interaction with Phospholipid Bilayers and Effect of Cholesterol
3. Discussion
4. Materials and Methods
4.1. Cav-2 Cloning
4.2. Cav-2 Expression in E. coli
4.3. Purification
4.3.1. Sephadex G75
4.3.2. Purification Using the His-Trap Affinity Column and Native Conditions
4.4. Preparation of SUVs and LUVs
4.5. Western Blotting
4.6. Circular Dichroism (CD)
4.7. Differential Scanning Calorimetry (DSC)
4.8. Fluorescence Intensity and Anisotropy Measurements
4.9. Prediction of Structural Information Obtained by Informatic Tools
4.10. Data Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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STEP | Protein Amount | Yield (%) |
---|---|---|
Whole lysate Periplasmic fraction Sephadex G75 Niquel Sepharose | 530.1 | 100 |
182.0 | 34.3 | |
147.0 | 27.7 | |
4.2 | 1.6 |
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Gorospe, B.; Moura, J.J.G.; Gutierrez-Merino, C.; Samhan-Arias, A.K. Biochemical and Biophysical Characterization of the Caveolin-2 Interaction with Membranes and Analysis of the Protein Structural Alteration by the Presence of Cholesterol. Int. J. Mol. Sci. 2022, 23, 15203. https://doi.org/10.3390/ijms232315203
Gorospe B, Moura JJG, Gutierrez-Merino C, Samhan-Arias AK. Biochemical and Biophysical Characterization of the Caveolin-2 Interaction with Membranes and Analysis of the Protein Structural Alteration by the Presence of Cholesterol. International Journal of Molecular Sciences. 2022; 23(23):15203. https://doi.org/10.3390/ijms232315203
Chicago/Turabian StyleGorospe, Berta, José J. G. Moura, Carlos Gutierrez-Merino, and Alejandro K. Samhan-Arias. 2022. "Biochemical and Biophysical Characterization of the Caveolin-2 Interaction with Membranes and Analysis of the Protein Structural Alteration by the Presence of Cholesterol" International Journal of Molecular Sciences 23, no. 23: 15203. https://doi.org/10.3390/ijms232315203