Direct Probing of Solvent Accessibility and Mobility at the Binding Interface of Polymerase (Dpo4)-DNA Complex

Abstract

Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined by the binding partners in nanospace but in many cases they are highly mobile and exchange with outside bulk solution. Here, we report our studies of the interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution. By systematic comparison of the interfacial water motions and local sidechain fluctuations in the apo, binary and ternary states of Dpo4, we observed that the DNA binding interface and active site is dynamically solvent accessible and the interfacial water dynamics are similar to the surface hydration water fluctuations on picosecond time scales. Our molecular dynamics simulations also show the binding interface full of water molecules and nonspecific weak interactions. Such a fluid binding interface facilitates the polymerase sliding on DNA for fast translocation while the spacious and mobile hydrated active site contributes to the low fidelity of the lesion-bypass Y-family DNA polymerase.

Recent Insight into the Kinetic Mechanisms and Conformational Dynamics of Y-Family DNA Polymerases

“… 129 The results from this study suggested that a hydrated binding interface facilitates the sliding of Dpo4 on the DNA substrate to allow for rapid translocation and that the dynamic solvent accessibility of the active site contributes to the low fidelity of Dpo4. 129 Additionally, the recent chemical shift backbone assignment of the polymerase core 130 and LF subdomain 131 of Dpo4 will facilitate the investigation of conformational dynamics at atomic-level resolution via protein NMR spectroscopy. Single-molecule FRET investigations have also begun to provide new insights into the kinetic mechanisms of several canonical DNA polymerases 132 − 137 and may prove to be useful in studying the Y-family DNA polymerases, as well.…”

Temporal nanofluidic confinements induce prebiotic condensation in water

“…The ndings can solve the water paradox in such a way that nano uidic effects in aqueous particle suspensions open up an abiotic route to biopolymerisation and polymer stabilization under chemical and thermodynamic conditions which are also prevalent within the crowded intracellular environment of living cells. The fact that polymerase enzymes also form temporal nanocon ned water clusters inside their active site 27,28 implies that the same physico-chemical effects are tapped for nucleotide condensation in water both by biochemical pathways and our reported abiotic route. These aspects indicate that our model is consistent with evolutionary conservatism stretching back to the era of prebiotic chemical evolution and the origin of cellular life 29 .…”

“…These aspects indicate that our model is consistent with evolutionary conservatism stretching back to the era of prebiotic chemical evolution and the origin of cellular life 29 . The consistency is further supported by the fact that water is not trapped by nanocon nements within the polymerase core but can exchange with the surrounding intracellular uid 28 -a situation that also exists in abiotic nanocon nements of water emerging temporarily between approaching particles in aqueous suspensions. Our experimental nding that under the reported conditions an amino acid catalyses the abiotic polymerisation of nucleotides may give a hint to a nano uidic origin of cooperation between amino acids and nucleotides evolving to the interdependent synthesis of proteins and nucleic acids in living cells 30 .…”

Mechanism‐Dependent Modulation of Ultrafast Interfacial Water Dynamics in Intrinsically Disordered Protein Complexes

“…To probe 5 orders of magnitude of solvation dynamics, we combined broadband femtosecond transient absorption (fsTA, ≤200 fs −2 ns) with time‐correlated single‐photon counting (TCSPC) to probe TDSS from ≤200 fs to 20 ns (Figures S8 and S9 and the Supporting Information). Broadband spectroscopy allowed us to obtain model‐free TDSS data bypassing the need for convolution‐based fitting of the kinetics at multiple wavelengths, which is typically used in solvation studies ,. Our analysis avoided an artificial smoothing from model‐based fitting and retained the experimental noise in the data.…”