Higher-Energy Collisional Dissociation Mass Spectral Networks for the Rapid, Semi-automated Characterization of Known and Unknown Ribonucleoside Modifications.

Higher-energy collisional dissociation (HCD) of modified ribonucleosides generates characteristic and highly reproducible nucleoside-specific tandem mass spectra (MS/MS). Here, we demonstrate the capability of HCD spectra in combination with spectral matching for the semi-automated characterization of ribonucleosides. This process involved the generation of an HCD spectral library and the establishment of a mass spectral network for rapid detection with high sensitivity and specificity in a retention time-independent fashion. Systematic spectral matching analysis of the MS/MS spectra of tRNA hydrolysates from different organisms has helped us to uncover evidence for the existence of novel ribonucleoside modifications such as s2Cm and OHyW-14. Such an untargeted label-free approach has the potential to be integrated with other methods, including those that use isotope labeling, to simplify the characterization of unknown modified ribonucleosides. These findings suggest the compilation of a universal spectral network, for the characterization of known and unknown ribonucleosides, could accelerate discoveries in the epitranscriptome.

Unfolding and Translocation of Knotted Proteins by Clp Biological Nanomachines: Synergistic Contribution of Primary Sequence and Topology Revealed by Molecular Dynamics Simulations.

We use Langevin dynamics simulations to model, at an atomistic resolution, how various natively knotted proteins are unfolded in repeated allosteric translocating cycles of the ClpY ATPase. We consider proteins representative of different topologies, from the simplest knot (trefoil 31), to the three-twist 52 knot, to the most complex stevedore, 61, knot. We harness the atomistic detail of the simulations to address aspects that have so far remained largely unexplored, such as sequence-dependent effects on the ruggedness of the landscape traversed during knot sliding. Our simulations reveal the combined effect on translocation of the knotted protein structure, i.e., backbone topology and geometry, and primary sequence, i.e., side chain size and interactions, and show that the latter can dominate translocation hindrance. In addition, we observe that due to the interplay between the knotted topology and intramolecular contacts the transmission of tension along the polypeptide chain occurs very differently from that of homopolymers. Finally, by considering native and non-native interactions, we examine how the disruption or formation of such contacts can affect the translocation processivity and concomitantly create multiple unfolding pathways with very different activation barriers.

Electrochemical Studies of Cation Condensation-Induced Collapse of Surface-Bound DNA.

In this paper, we demonstrate the ability to control and electrochemically monitor nucleic acid conformation by inducing collapse of short, surface-bound nucleotides (7-28 nucleotides). More specifically, we monitored changes in a 5'-electrode-bound DNA structure via changes in the faradaic current related to the reduction/oxidation of a 3'-terminal-appended redox molecule. Reversible DNA collapse was induced by cation condensation achieved by either reducing the dielectric permittivity of the interrogation solution or by the addition of multivalent cations such as the polyamine spermidine (3+). Additionally, we find that while the change in electrochemical signal associated with surface bound DNA collapse is dependent on nucleic acid length and surface packing density, the solution conditions (e.g., dielectric permittivity) required for collapse remain constant. As such, we find that collapse of the short DNA strands occurs when the effective charge of the DNA backbone is ∼73-89% neutralized by cations in solution/buffer, according to Manning's theory on cation condensation. This work provides new insight into the structure function relationship of surface-bound nucleic acids and how this is manifested in electrochemical signaling.

Density functional tight-binding and infrequent metadynamics can capture entropic effects in intramolecular hydrogen transfer reactions.

Infrequent metadynamics uses biased simulations to estimate the unbiased kinetics of a system, facilitating the calculation of rates and barriers. Here the method is applied to study intramolecular hydrogen transfer reactions involving peroxy radicals, a class of reactions that is challenging to model due to the entropic contributions of the formation of ring structures in the transition state. Using the self-consistent charge density-functional based tight-binding (DFTB) method, we applied infrequent metadynamics to the study of four intramolecular H-transfer reactions, demonstrating that the method can qualitatively reproduce these high entropic contributions, as observed in experiments and those predicted by transition state theory modeled by higher levels of theory. We also show that infrequent metadynamics and DFTB are successful in describing the relationship between transition state ring size and kinetic coefficients (e.g., activation energies and the pre-exponential factors).

Assessing Generic Collective Variables for Determining Reaction Rates in Metadynamics Simulations.

A persistent challenge in using the metadynamics method is deciding which degrees of freedom, or collective variables, should be biased because these selections are not obvious and require intuition about the system being studied. There are, however, collective variables, which can be constructed with only basic knowledge about the system studied, that provide an opportunity to alleviate this issue. We simulated two different reacting systems where two types of such collective variables (SPRINT coordinates and the collective variable-driven hyperdynamics method) were biased following the infrequent metadynamics method in order to recover the rates of reactions. We demonstrate that both generic collective variables are capable of reproducing the reaction rates of both systems and can enhance the efficiency of the simulation when compared to typical collective variables.

Benchmarking Density Functional Based Tight-Binding for Silver and Gold Materials: From Small Clusters to Bulk.

We benchmark existing and improved self-consistent-charge density functional based tight-binding (SCC-DFTB) parameters for silver and gold clusters as well as for bulk materials. In the former case, our benchmarks focus on both the structural and energetic properties of small-size AgN and AuN clusters (N from 2 to 13), medium-size clusters with N = 20 and 55, and finally larger nanoparticles with N = 147, 309, and 561. For bulk materials, structural, energetics and elastic properties are discussed. We show that SCC-DFTB is quite satisfactory in reproducing essential differences between silver and gold aggregates, in particular their 2D-3D structural transitions, and their dependency upon cluster charge. SCC-DFTB is also in agreement with DFT and experiments in the medium-size regime regarding the energetic ordering of the different low-energy isomers and allows for an overall satisfactory treatment of bulk properties. A consistent convergence between the cohesive energies of the largest investigated nanoparticles and the bulk's is obtained. On the basis of our results for nanoparticles of increasing size, a two-parameter analytical extrapolation of the cohesive energy is proposed. This formula takes into account the reduction of the cohesive energy for undercoordinated surface sites and converges properly to the bulk cohesive energy. Values for the surface sites cohesive energies are also proposed.