REDISTRIBUTION OF STATES AND INDUCING NEW CHANNELS FOR CONFORMATIONAL CHANGE: COMPUTATIONAL STUDIES ON CALMODULIN
Ayşe Özlem Aykut
Material Science and Engineering, PhD Dissertation, 2013
Prof. Dr. Canan Atılgan (Thesis Supervisor), Prof. Dr. Ali Rana Atılgan (Thesis Co-Supervisor), Asst. Prof. Deniz Sezer, Asst. Prof. Elif Özkırımlı, Prof. Dr. Pemra Doruker.
Date &Time: June 10th, 2013 - 10:30
Place: FENS G032
Keywords : Molecular-Dynamics Simulations; Calcium-Loaded Calmodulin; Central Helix; Proteins; Spectroscopy; Algorithm; pH; Plasticity; Elastic Network Models; Interdomain Interactions; Macromolecular Motions; Multidimensional NMR
In vitro experiments demonstrate that large conformational changes in many proteins are observed as “rare events” occurring in microseconds timescales. For proteins that sustain a plethora of functions, it is imperative that different conformational states be achieved readily under slightly differing environmental conditions in vivo. We investigate how perturbations that may be experienced by proteins in their fluctuating environments may be invoked to facilitate their access to different micro states, using the example of calcium loaded calmodulin (Ca2+-CaM). As perturbation, we introduce mutation of a single residue or protonation of group of residues on Ca2+-CaM. After performing molecular dynamics (MD) simulations on the perturbed systems, we observe distinct conformational changes within tens of nanoseconds, that otherwise occur on the time scales of microseconds. In particular, a reversible change between the extended and compact Ca2+-CaM structure may be invoked via the E31A mutation. This compact form bears a bent linker which is observed in many of the ligand bound forms of Ca2+-CaM. Protonation of ten acidic residues also leads to a large conformational change in less than 100 ns. The structure attained is compact and does not have a bent in the linker, however is consistent with fluorescence resonance energy transfer experimental results. It is compatible with structures from an ensemble NMR data. Analysis of the MD trajectories implies that the key events leading to the conformational change by protonation of ten residues begins with a formation of a salt bridge between the N-lobe and the linker, followed by the bending of the C-lobe and the organization of a stabilizing hydrophobic patch between the lobes. Barrier crossing between extended and compact forms of CaM which is normally a rare event is facilitated by protonation of high pK(a) residues by shielding the repulsive electrostatic interactions between the two lobes. Protonation of ten residues on Ca2+-CaM mimics the pH 5 environment and can be utilized to show the impact of pH changes in the cell to conformational change.
While studying the dynamics of Ca2+-CaM under different perturbation scenarios, we find that the number of ions reside in close proximity to the N terminal domain (NTD) and C terminal domain (CTD) are depleted and the linker fluctuations are rigidified in the high ionic strength (IS) environment. Moreover, the dynamics of the system in the high IS environment is slowed down due to the screening effect of the ions between the domains.
Application of external perturbations to extended Ca2+-CaM in the form of forces vs. displacements yield to complementary results. We find that both approaches designate the same two regions on the protein structure making these regions potential sites for manipulation to yield expected conformational change. Local force perturbation implicates charged residues while local displacement perturbation finds polar and hydrophobic residues in these two regions which reflect the differences inherent in the thermodynamic functions optimized by two approaches.