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Here, we introduce a framework that combines instantaneous and temporal graph analysis of hydrogen\u2010bond networks derived from molecular dynamics (MD) trajectories to quantify ligand\u2010directed hydrogen\u2010bond connectivity. We apply the method to\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>E. coli<\/jats:italic>\n                    <\/jats:styled-content>\n                    dihydrofolate reductase (DHFR) and its L28R mutant, computing shortest hydrogen\u2010bond paths from all residues to the substrate dihydrofolate (DHF). The instantaneous analysis reveals that DHF\u2010directed connectivity is organized through a sparse set of preferred routes, with D27 and T113 acting as prominent hubs in the wild\u2010type enzyme. Temporal analysis highlights residues that preferentially support time\u2010ordered DHF\u2010directed connectivity. Comparison with L28R shows that the mutation preserves the main substrate\u2010contacting architecture and the overall communication scaffold but redistributes pathway usage, persistence, and temporal convergence. The network\u2010derived hotspots partially overlap with independent coevolution signals, most strongly in the K109\u2013I115 region, while overlap with cryptic\u2010site predictors is more limited. This pattern indicates that the hydrogen\u2010bond network captures evolutionarily supported communication regions in DHFR that are not fully recovered by static structural approaches. The framework is broadly applicable to ligand\u2010binding proteins and provides a route to identify persistent, delayed, and mutation\u2010sensitive signaling pathways directly from time\u2010ordered simulation data.\n                  <\/jats:p>","DOI":"10.1002\/prot.70182","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T05:29:06Z","timestamp":1790054946000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Temporal Hydrogen\u2010Bond Network Analysis Reveals Substrate\u2010Directed Connectivity in Dihydrofolate Reductase"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2516-1922","authenticated-orcid":false,"given":"Tandac F.","family":"Guclu","sequence":"first","affiliation":[{"name":"Vocational School of Health Services Istinye University  Istanbul 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