The basic biochemical phenomenon, protein unfolding, has recently seen a growing interest. Understanding protein unfolding from the kinetic and mechanistic point of view is essential for optimizing protein properties, like stability or solubility, utilizing methods of protein engineering. This bachelor thesis aims to analyze the unfolding of haloalkane dehalogenases LinB wild type and stabilized variant LinB116 and compare it with DhaA wild type and stabilized variant DhaA115. Comparison of stable and unstable variants can show us the effect of stabilizing mutations on overall protein resilience to high temperatures. The DhaA and LinB unfolding mechanisms will be studied using enhanced sampling molecular dynamics simulations and by the construction of Markov models of observed macrostates.
Thesis goals:
1. Literature review regarding protein unfolding, aggregation, and model proteins haloalkane dehalogenases and description of methods regarding homology modelling, molecular dynamics simulations, adaptive sampling, and the identification of aggregation-prone regions,
2. Molecular dynamics simulation of the temperature-related unfolding of haloalkane dehalogenase LinB and stable variant LinB116,
3. Analysis of the simulations using Markov state models and comparison with the haloalkane dehalogenase DhaA and its stabilized variant DhaA115,
4. Identification of aggregation-prone regions,
5. Discussion of the outcome with literature or available experimental results.
Literature:
A. M. Lesk, Introduction to protein architecture: The Structural Biology of Proteins. Oxford Univ. Press 2003
A. Hospital, J. R. Goñi, M. Orozco, and J. L. Gelpí, “Molecular dynamics simulations: Advances and applications,” Adv. Appl. Bioinforma. Chem., vol. 8, no. 1, pp. 37–47, 2015, doi: 10.2147/AABC.S70333.
V. Daggett, “Molecular dynamics simulations of the protein unfolding/folding reaction,” Acc. Chem. Res., vol. 35, no. 6, pp. 422–429, 2002, doi: 10.1021/ar0100834.
Y. I. Yang, Q. Shao, J. Zhang, L. Yang, and Y. Q. Gao, “Enhanced sampling in molecular dynamics,” J. Chem. Phys., vol. 151, no. 7, 2019, doi: 10.1063/1.5109531.
A. Kuriata, V. Iglesias, J. Pujols, M. Kurcinski, S. Kmiecik, and S. Ventura, “Aggrescan3D (A3D) 2.0: Prediction and engineering of protein solubility,” Nucleic Acids Res., vol. 47, no. W1, pp. W300–W307, 2019, doi: 10.1093/nar/gkz321.
T. Koudelakova et al., “Haloalkane dehalogenases: Biotechnological applications,” Biotechnol. J., vol. 8, no. 1, pp. 32–45, 2013, doi: 10.1002/biot.201100486.