Destabilization of a prion protein (PrP) induces a conformational change that alters a normal prion protein (PrPC) to an abnormal prion protein (PrPSC). The structural stability of PrP is dramatically destabilized by unfavorable external environmental conditions and mutations in the protein. Environmental pH is a critical determinant of misfolding and aggregation of PrP, which leads to fatal neurodegenerative diseases. Because protonation of H187 and mutation of the hydrophobic core on the H2-H3 bundle are strongly linked to a change in PrP stability, I examined its charged residues R156, E196, and D202 around H187 and hydrophobic residues V176, V180, T183, V210, I215, and Y218. Interestingly, there are reports on mutants, such as V176G, T183A, H187R, E196A, D202N, I215V, and Y218N, which cause genetic prion diseases. First, I focused on the mechanism by which an acidic pH and mutants disrupt this electrostatic network and how this broken network destabilizes the PrP structure. Towards this objective, I performed a temperature-based replica-exchange molecular dynamics (T-REMD) simulation using a cumulative 252μs simulation time. I measured the distance between amino acids comprising four salt bridges (R156–E196/D202 and H187–E196/D202). Our results showed that the spatial configuration of the electrostatic network was significantly altered by an acidic pH and mutations. The structural alteration in the electrostatic network increased the RMSF value around the first helix (H1). Thus, the structural stability of H1 anchored to the H2–H3 bundle was decreased, which induced separation of R156 from the electrostatic network. Analysis of the anchoring energy also showed that the two salt-bridges (R156-E196/D202) are critical for PrP stability. Second, I focused on the hydrophobic interaction. Not only electrostatic interaction but also hydrophobic interaction is the main driving force for protein folding, critically affecting the stability and solubility of the protein. To examine the importance of the hydrophobic core in the PrP, I chose six amino acids (V176, V180, T183, V210, I215, and Y218) that form the hydrophobic core at the middle of the H2-H3 bundle. A few pathological mutants of these amino acids have been reported, such as V176G, V180I, T183A, V210I, I215V, and Y218N. I revealed how these mutations affect the hydrophobic core and thermostability of PrP. Towards this, I used a temperature-based replica-exchange molecular dynamics (T-REMD) simulation for extensive ensemble sampling. From the T-REMD ensemble, I calculated the protein folding free energy difference between wild-type and mutant PrP using the thermodynamic integration (TI) method. Our results showed that the mutants V176G, T183A, I215V, and Y218N decreased PrP stability. At the atomic level, I examined the change [valine-valine to valine-isoleucine (and vice versa)] in pair-wise hydrophobic interactions, which is induced by mutation V180I, V210I (I215V) at the 180th–210th (176th–215th) pair. Additionally, I investigated the importance of the π-stacking between Y218 and F175.
Table Of Contents
Ⅰ. Introduction 1 1.1) Prion protein and neurodegenerative diseases 1 1.2.1) Electrostatic network in prion protein 1 1.2.2) Extensive conformational ensemble sampling with temperature-based replica-exchange molecular dynamics (T-REMD) 4 1.3.1) Hydrophobic interaction in prion protein 5 1.3.2) Free energy calculation with thermodynamic integration (TI) 7 Ⅱ. Results 10 2.1) Electrostatic network in PrP 10 2.1.1) The spatial configuration of wild-type PrP under neutral pH conditions 10 2.1.2) Environmental pH conditions affect the electrostatic network 13 2.1.3) The protonated histidine (Hp187) critically affects the thermostability of the E196A mutant 19 2.1.4) D202N mutant induces a weakly anchored conformation 24 2.1.5) The separation of alanine (R156A) from the electrostatic network 29 2.2) Hydrophobic core in PrP 35 2.2.1) V180I mutant could destabilize the glycan structure 35 2.2.2) T183A mutant critically destabilizes the hydrophobic core in PrP 37 2.2.3) Hydrophobic interactions in PrPV180I and PrPT183A 38 2.2.4) Loss of hydrophobic interaction in V176G mutant 41 2.2.5) Mutation effect of V210I and the interaction with P158, V180, and T183 43 2.2.6) Spatial structures enforce isoleucine at the 215th position 47 2.2.7) The effect of disappearing of π-stacking in Y218N mutant 49 Ⅲ. Discussion 51 Ⅳ.Materials and methods 58 4.1) Temperature-based replica-exchange molecular dynamics (T-REMD) 58 4.2) Thermodynamic integration (TI) 60 4.3) Defining unfolding states 61 Reference 63 요약문 71