Significance: The cardiac thin filament (CTF) is a complex and highly dynamic multiprotein complex that regulates heart function at the molecular level. Mutations in proteins that comprise the thin filament cause cardiomyopathic remodeling that is due, in part, to dysregulation of Ca ²⁺ binding. Many of the known mutations in multiple thin filament proteins occur at a significant physical distance from the Ca ²⁺ binding domain; thus, the elucidation of molecular mechanisms has been challenging. We have developed and now apply an atomistic model of the CTF to this question and have obtained molecular insight that both provides disease mechanisms and facilitates future rational drug design. Finding the molecular cause of disease is the first step in designing molecular interventions.

The relevance of sub-picosecond protein motions to the catalytic event remains a topic of debate. Heavy enzymes (isotopically substituted) provide an experimental tool for bond-vibrational links to enzyme catalysis. A recent transition path sampling study with heavy purine nucleoside phosphorylase (PNP) characterized the experimentally observed mass-dependent slowing of barrier crossing (Antoniou, D.; Ge, X.; Schramm, V. L.; Schwartz, S. D. J. Phys. Chem. Lett.2012, 3, 3538). Here we computationally identify second-sphere amino acid residues predicted to influence the freedom of the catalytic site vibrational modes linked to heavy enzyme effects in PNP. We mutated heavy and light PNPs to increase the catalytic site vibrational freedom. Enzymatic barrier-crossing rates were converted from mass-dependent to mass-independent as a result of the mutations. The mutagenic uncoupling of femtosecond motions between catalytic site groups and reactants decreased transition state barrier crossing by 2 orders of magnitude, an indication of the femtosecond dynamic contributions to catalysis.