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In the 2022 publication, Cao et al. designed sets of de novo minibinders intended to bind to selected interfaces on various natural target proteins. The study focused on the method used to computationally design and improve these proteins, yielding their pre-optimized (pOpt) and optimized (Opt) sequences, respectively. Although many static metrics were used to analyze differences between the Opt and pOpt minibinders and to evaluate the success of the optimization method, molecular dynamics were not included in these analyses. This work builds upon Cao et al., measuring residue-level flexibility via root-mean-squared fluctuation (RMSF) and high-dimensional movement variance via principal component analysis (PCA) from molecular dynamics (MD) simulations, providing a new lens for analyzing these de novo minibinders by incorporating their dynamics. Here, we identify a relationship between the computational optimization and the binding-induced rigidification of the de novo minibinders. Our results indicate a consistent rigidification of the Opt minibinders upon binding, particularly at residues within secondary structures that form the binding interface. While global analysis of RMSF shows high variance, local analysis of the interface revealed a significant \"locking\" effect in VirB8 and CD3d Opt minibinder variants upon binding, suggesting higher interfacial complementarity than in their respective pOpt minibinders. However, the high variance within the results and the case of the TrkA minibinders reveal a more nuanced dynamic profile. These findings necessitate the inclusion of molecular dynamics in the de novo design pipeline to provide a more holistic approach to predicting and determining success.</p>"},{"label":"Digital Identifier","value":"b2026-kermath"},{"label":"Digital Object Identifier (DOI)","value":"https://doi.org/10.14418/6qzdogmt"},{"label":"Digital Origin","value":["born digital"]},{"label":"Discipline","value":["Biology"]},{"label":"Date","value":"April 15, 2026"},{"label":"EDTF Date","value":"D:00 M:00 Y:2026"},{"label":"File Format","value":["application/pdf"]},{"label":"Genre","value":["theses"]},{"label":"Group","value":["Theses & Dissertations"]},{"label":"Held By","value":"Wesleyan University Library, Wesleyan University, Middletown, CT, USA"},{"label":"Language","value":["English"]},{"label":"Repository","value":["University Archives"]},{"label":"Rights","value":["In Copyright"]},{"label":"Thesis Advisor(s)","value":["Smith, Colin 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