RESUMO
The influence of ß-lactoglobulin (ßLG) on the fast sub-picosecond collective hydration dynamics in the solvent was investigated by THz absorption spectroscopy as a function of pH. It is well-known that a change in pH from pH 6 to pH 8 reversibly opens or closes the binding cavity by a transition of the E-F loop. Furthermore, the aggregation of the protein into dimers is affected, which is thought to be triggered by changes in the enzyme's electrostatic potential. Our data reveal that pH has a clear influence on the THz absorption of ßLG. We discuss this influence in light of the changes observed in the sub-psec solute/solvent dynamics when probed by THz spectroscopy, which are, in turn, seen to correlate with changes in the pH value.
Assuntos
Lactoglobulinas/química , Animais , Bovinos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Solventes , Eletricidade Estática , Espectroscopia Terahertz , Água/químicaRESUMO
The main focus of enzymology is on the enzyme rates, substrate structures, and reactivity, whereas the role of solvent dynamics in mediating the biological reaction is often left aside owing to its complex molecular behavior. We used integrated X-ray- and terahertz- based time-resolved spectroscopic tools to study protein-water dynamics during proteolysis of collagen-like substrates by a matrix metalloproteinase. We show equilibration of structural kinetic transitions in the millisecond timescale during degradation of the two model substrates collagen and gelatin, which have different supersecondary structure and flexibility. Unexpectedly, the detected changes in collective enzyme-substrate-water-coupled motions persisted well beyond steady state for both substrates while displaying substrate-specific behaviors. Molecular dynamics simulations further showed that a hydration funnel (i.e., a gradient in retardation of hydrogen bond (HB) dynamics toward the active site) is substrate-dependent, exhibiting a steeper gradient for the more complex enzyme-collagen system. The long-lasting changes in protein-water dynamics reflect a collection of local energetic equilibrium states specifically formed during substrate conversion. Thus, the observed long-lasting water dynamics contribute to the net enzyme reactivity, impacting substrate binding, positional catalysis, and product release.