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Source URL: https://webapps.wesleyan.edu/grad_thesis/thesis_file/3262
Original filename: Hwang_Pete_Item_39_Copies.pdf"},{"label":"Author-supplied Keywords","value":["StACKER","Pi-stacking","Molecular dynamics","Ribosome","Decoding center","mRNA translation","System stacking fingerprints","Pairwise stacking fingerprints","Ribonucleoprotein dynamics","Amino acids","Principal component analysis","K-means clustering"]},{"label":"Degree","value":["Master of Arts"]},{"label":"Description","value":"

Biomolecular function often depends on dynamic interaction networks that are difficult to capture using static structures alone. In the ribosome decoding-center neighborhood, local sequence changes can alter interactions among rRNA, mRNA, tRNA, and nearby proteins, potentially shifting the system between distinct conformational states associated with different translational behaviors (Abeyrathne et al., 2016; Scopino et al., 2020; Sun et al., 2024). StACKER (Stacking Analysis and Conformational Kinetics for Examining Residues) is a Python framework for quantifying nucleotide π-stacking in molecular dynamics trajectories through System Stacking Fingerprints (SSFs) and Pairwise Stacking Fingerprints (PSFs) (Sakkas, 2025). In its original form, however, StACKER was limited to nucleotide-only analysis. This thesis refers to this version of the implementation as StACKER 1.0. This thesis presents StACKER 2.0, a significantly expanded stacking-fingerprint framework that incorporates aromatic amino acids and arginine into the analysis of mixed RNA-protein systems. New default center-of-geometry definitions were introduced for pyrimidines, purines, aromatic amino-acid side chains, and the arginine guanidinium group. A new distance cutoff for amino-acid-involving stacking interactions was established using blinded visual classification in the PyMOL structure visualization tool and implemented PSF generation for amino-acid-containing residue pairs. In parallel, the computational performance of SSF generation was substantially improved through a redesigned and parallelized distance-calculation pipeline. StACKER 2.0 was applied to molecular dynamics trajectories of a ribosome decoding-center subsystem differing in A-site and +1 codon identity. In the benchmark comparison between tGGG_aCCU_+1GCU and tGGG_aCCU_+1CGU, the updated framework preserved the major systems-level distinction previously observed in StACKER 1.0 while revealing new top-changing residue pairs that included amino-acid–amino-acid and nucleotide–amino-acid interactions (Sakkas, 2025). Pairwise stacking fingerprints showed that these newly identified pairs exhibit distinct codon-dependent geometric distributions, demonstrating that local structural changes extend beyond nucleotide-only contacts. K-means clustering and principal component analysis of frame-wise SSFs further showed that tGGG_aCCU_+1GCU occupies a distinct region of stacking-fingerprint space relative to the other trajectories, supporting the interpretation that the expanded stacking representation captures a meaningful systems-level conformational state. Together, these results show that extending stacking analysis to include amino acids improves the descriptive reach of the StACKER framework while preserving its original interpretability. StACKER 2.0 therefore provides a more chemically complete approach for analyzing dynamic interaction networks in ribonucleoprotein systems and establishes a foundation for future systems-level studies of codon-sensitive ribosome behavior.

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