Abstract
Protein synthesis relies on accurate mRNA decoding by tRNA, a process guided by EF-Tu. We investigated how mutations at a conserved residue, aspartate 81, affect EF-Tu function using GTPase assays, AlphaFold modeling, and quantum-sensing-based super-resolution force spectroscopy (SURFS). All D81 variants retained GTPase activity but impaired tRNA release, revealed by sub-nucleotide ribosome footprinting. AlphaFold3 modeling suggests that D81 mutations disrupt magnesium coordination and interaction with the sarcin-ricin loop in the GTP-bound state. AlphaFold2-based sequence-structure analysis indicates that D81 anchors coevolutionary constraints, and its mutation enables cryptic structural variation. These results show how a single conserved residue links catalytic coordination, allosteric communication, and evolutionary constraint, offering mechanistic insight into translation fidelity and demonstrating the utility of an unconventional force spectroscopy in probing ribosome dynamics.