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    Exploring functional dynamıcs of bacterial and human ribosome structures via coarse grained techniques
    (Fen Bilimleri Enstitüsü, 2020) Güzel, Pelin ; Kürkçüoğlu Levitas, Ayşe Özge ; 638208 ; Kimya Mühendisliği Ana Bilim Dalı
    The ribosome is a molecular machine that catalyzes protein synthesis in three kingdoms of life. This process is regulated by the binding of several protein factors to the main ribosomal complex core during different steps of translation, which are initiation, elongation, termination and recycling. This thesis employs coarse-grained (CG) computational techniques that focuses on differences/similarities in especially dynamical behavior between bacterial and human ribosomal complexes at different stages of translation. Investigating the allosteric communication pathways between distant functional sites of the molecular machine is the other focus. In a CG model, sets of atoms are represented by pseudo-atoms to decrease the degrees of freedom and simulation cpu time. In this thesis, residue interaction network, CG molecular dynamics (MD) simulations and anisotropic network model (ANM), which all use reduced representations of the structures based on one-bead coarse-graining, are employed. The findings of at least 500 ns CGMD simulations and ANM using normal mode analysis are interpreted to get an insight into the overall dynamical behavior of ribosomal complexes. Both methods give consistent results with experimental data on the ribosomal complexes. In order to map allosteric communications in the complexes, perturbation response scanning (PRS) and k-shortest pathways calculations are performed using the CGMD trajectories and the residue network model, respectively. PRS provides information on the influence or effectiveness of a given residue in signal transmission. k-shortest pathways calculations requires a source and a sink node and calculates suboptimal pathways. k-shortest path technique is applied to residue interaction networks. CGMD calculations are performed by the RedMD software. ANM using normal mode analysis, residue interaction network model and k-shortest pathways calculations are performed by in-house codes. CGMD calculations are performed on T. thermophilus, E. coli and H. sapiens ribosomal structures. Comparison of experimental and theoretical B-factors (both calculated from CGMD and ANM) shows that the findings are in accord with experimental data. Correlation coefficients are especially in an acceptable range (0.60-0.70) for rRNA portions between experimental-CGMD findings. Most fluctuating parts are found at the solvent-exposed sites on both 30S (small subunit) and 50S (large subunit). For the bacterial ribosome case; head, beak and spur of 30S are found as highly flexible parts. Additionally, some of the sites around the neck where tRNAs and mRNA bind to the ribosome are also flexible. Results point to two flexible fragments in the 50S. One is the L1 stalk which is composed of the uL1 protein and helices H76-H78 of the 23S rRNA. The release of the E-tRNA requires the correct positioning of L1 stalk. Additionally, L7/L12 stalk is another high fluctuating part of the 50S. It is known to make an anticorrelated movement with the L1 stalk. These lateral stalks are found to play a highly crucial role in the proper functioning of the ribosome. The analysis of E. coli crystal structure fluctuations has also shown that the protein components in the ribosomal complex are also in accord with experimental studies. Dynamical cross-correlation maps (DCCMs) based on CGMD trajectories are generated. The findings indicate that there is more coupling within a subunit than between. Coupling within protein chains is also higher than coupling within rRNA chains indicating the more compact globular structures of proteins than rRNAs. Global dynamics of ribosomal complexes are analyzed by employing ANM using normal mode analysis. The calculations are based on four T. thermophilus and one H. sapiens crystal structure. The characteristic conformational changes; ratchet-like rotation (both in T. thermophilus and H. sapiens), 30S/40S head rotation (both), anti-correlated movement of L1 and L7/L12 stalks (T. thermophilus), 40S head and beak regions towards E-tRNA (H. sapiens and T. thermophilus with PDB ID:4v9h), 40S body rotation around vertical axis perpendicular to classical rotation axis called as "subunit rolling" (H. sapiens) are observed. In the second part of this thesis, allosteric regulation mechanisms in the ribosomal complexes are investigated and compared for bacteria and human. For this purpose, k-shortest pathways algorithms and PRS calculations are employed. k-shortest pathway algorithms (Yen's and Dijkstra) are applied to the networks generated from residue interaction. Potential allosteric communication pathways between DC-PTC and the ribosomal tunnel-PTC (peptidyl transferase center) are explored by these CG techniques. Additionally, residues found on the shortest pathways are analyzed in terms of rigidity/flexibility (according to deformation energy analysis) and being "hub" residue or not (according to "contact number" analysis). The calculated suboptimal pathways between the tunnel and the PTC in T. thermophilus ribosomal structures agree with the previously proposed allosteric pathways. Motivated by the success of these models in the tunnel-PTC case, they are employed for investigating potential allosteric communication pathways between highly distant functional sites, the DC and the PTC of the ribosome, which are known to communicate during the translation process. The analysis suggests that especially B3 and B2a inter-subunit bridges are critical for the long-range signal transmission between the DC and the PTC. Then the potential allosteric communication pathways are also investigated on the human ribosomal complex for the first time to our knowledge. The human ribosomal complex seems to employ similar suboptimal pathways between the tunnel and the PTC as well as the DC and the PTC. In this line, B3 and B2a are highlighted as critical hubs in the long-range signal transmission in the ribosomal structures and can be evaluated as drug binding sites. Then, the ribosomal tunnel where the growing polypeptide chain passes through before emerging at the solvent is studied in more detail. In the bacterial ribosome, the ribosomal exit tunnel walls are formed by the 23S rRNA, uL4, uL22 and a bacteria-specific extension of uL23. In eukaryotes, the bacteria-specific extension of uL23 overlaps with eL39. uL4 and uL22 form a constriction within the tunnel which is located approximately 30 Å from the PTC. An additional loop in uL4 makes this constriction narrower in eukaryotes than prokaryotes. In this line, the ribosomal tunnels of both T. thermophilus and H. sapiens are extracted and analyzed in detail. The additional loop in uL4 in eukaryotes changes the dynamical behavior of this region, which may be related to the macrolide binding discrimination in both structures. To get a deeper understanding, the PRS is applied to the conserved critical residues in uL4 which are located at the constriction region of the tunnel. In the same chapter of the thesis, the allosteric communication in the ribosomal exit tunnel is also discussed focusing on both nascent chain interactions and the trigger factor (TF) recruitment mechanism induced by the ribosomal tunnel on E. coli ribosomal structure. PRS analysis is carried out to identify effectors/sensors around the tunnel. The findings are consistent with experimental observations indicating the allosteric communications between PTC-ribosomal tunnel and chaperone binding site in uL23-lower part of ribosomal tunnel. Especially, uL23 can be considered as a novel drug designing targeting its non-conserved pocket.