Alexander V. Predeus

Conformational sampling of peptides in the presence of protein crowders from AA/CG-multiscale simulations.

Macromolecular crowding is recognized as an important factor influencing folding and conformational dynamics of proteins and nucleic acids. Previous views of crowding have focused on the mostly entropic volume exclusion effect of crowding, but recent studies are indicating the importance of enthalpic effects, in particular, changes in electrostatic interactions due to crowding. Here, temperature replica exchange molecular dynamics simulations of trp-cage and melittin in the presence of explicit protein crowders are presented to further examine the effect of protein crowders on peptide dynamics. The simulations involve a three-component multiscale modeling scheme where the peptides are represented at an atomistic level, the crowder proteins at a coarse-grained level, and the surrounding aqueous solvent as implicit solvent. This scheme optimally balances a physically realistic description for the peptide with computational efficiency. The multiscale simulations were compared with simulations of the same peptides in different dielectric environments with dielectric constants ranging from 5 to 80. It is found that the sampling in the presence of the crowders resembles sampling with reduced dielectric constants between 10 and 40. Furthermore, diverse conformational ensembles are generated in the presence of crowders including partially unfolded states for trp-cage. These findings emphasize the importance of enthalpic interactions over volume exclusion effects in describing the effects of cellular crowding.

Optically active calixarenes conduced by methylene substitution

The first method for the synthesis of optically active calix[4]arenes that are chiral as a result of substitution on the methylene bridges is described. The key step in the synthesis involves the reaction of a biscarbene complex with a diyne, which generates two of the benzene rings and the macrocyclic ring of the calix in a single transformation. The utility of this triple annulation process is demonstrated in the synthesis of di- and tetramethoxycalix[4]arenes. The flexibility of this synthetic approach is demonstrated by the synthesis of two diastereomers of the tetramethoxycalix[4]arenes in which each is synthesized in a stereoselective fashion by proper control of the absolute configurations of the methoxy groups in the biscarbene complex and in the diyne.

DNA bending propensity in the presence of base mismatches: Implications for DNA repair

DNA bending is believed to facilitate the initial recognition of the mismatched base for repair. The repair efficiencies are dependent on both the mismatch type and neighboring nucleotide sequence. We have studied bending of several DNA duplexes containing canonical matches: A:T and G:C; various mismatches: A:A, A:C, G:A, G:G, G:T, C:C, C:T, and T:T; and a bis-abasic site: X:X. Free-energy profiles were generated for DNA bending using umbrella sampling. The highest energetic cost associated with DNA bending is observed for canonical matches while bending free energies are lower in the presence of mismatches, with the lowest value for the abasic site. In all of the sequences, DNA duplexes bend toward the major groove with widening of the minor groove. For homoduplexes, DNA bending is observed to occur via smooth deformations, whereas for heteroduplexes, kinks are observed at the mismatch site during strong bending. In general, pyrimidine:pyrimidine mismatches are the most destabilizing, while purine:purine mismatches lead to intermediate destabilization, and purine:pyrimidine mismatches are the least destabilizing. The ease of bending is partially correlated with the binding affinity of MutS to the mismatch pairs and subsequent repair efficiencies, indicating that intrinsic DNA bending propensities are a key factor of mismatch recognition.

Energetic and Structural Details of the Trigger-Loop Closing Transition in RNA Polymerase II

An evolutionarily conserved element in RNA polymerase II, the trigger loop (TL), has been suggested to play an important role in the elongation rate, fidelity of selection of the matched nucleoside triphosphate (NTP), catalysis of transcription elongation, and translocation in both eukaryotes and prokaryotes. In response to NTP binding, the TL undergoes large conformational changes to switch between distinct open and closed states to tighten the active site and avail catalysis. A computational strategy for characterizing the conformational transition pathway is presented to bridge the open and closed states of the TL. Information from a large number of independent all-atom molecular dynamics trajectories from Hamiltonian replica exchange and targeted molecular dynamics simulations is gathered together to assemble a connectivity map of the conformational transition. The results show that with a cognate NTP, TL closing should be a spontaneous process. One major intermediate state is identified along the conformational transition pathway, and the key structural features are characterized. The complete pathway from the open TL to the closed TL provides a clear picture of the TL closing.

Differential Mismatch Recognition Specificities of Eukaryotic MutS Homologs, MutSα and MutSβ

In eukaryotes, the recognition of the DNA postreplication errors and initiation of the mismatch repair is carried out by two MutS homologs: MutSα and MutSβ. MutSα recognizes base mismatches and 1 to 2 unpaired nucleotides whereas MutSβ recognizes longer insertion-deletion loops (IDLs) with 1 to 15 unpaired nucleotides as well as certain mismatches. Results from molecular dynamics simulations of native MutSβ:IDL-containing DNA and MutSα:mismatch DNA complexes as well as complexes with swapped DNA substrates provide mechanistic insight into how the differential substrate specificities are achieved by MutSα and MutSβ, respectively. Our simulations results suggest more extensive interactions between MutSβ and IDL-DNA and between MutSα and mismatch-containing DNA that suggest corresponding differences in stability. Furthermore, our simulations suggest more expanded mechanistic details involving a different degree of bending when DNA is bound to either MutSα or MutSβ and a more likely opening of the clamp domains when noncognate substrates are bound. The simulation results also provide detailed information on key residues in MutSβ and MutSα that are likely involved in recognizing IDL-DNA and mismatch-containing DNA, respectively.

Rational synthesis for all all-homocalixarenes.

Calixarenes and related compounds are established scaffolds useful as ligands and very popular as building blocks for molecular design. At the same time, the use of these compounds is virtually always defined by the ability of researchers to prepare them efficiently in useful quantities, and in a controlled fashion. As an example, calix[4]arenes, calix[6]arenes, and calix[8]arenes gained popularity very quickly after Gutsche et al. developed and perfected onestep methods for their preparation. Fitting geometry, namely appropriate ring size and dominant conformation, are of paramount importance in most macrocycle applications, yet the overwhelming majority of publications still only report modifications of the three above-mentioned easily prepared calixarene cores. Homocalixarenes have been a subject of scientific interest for a couple of decades. Whereas calixarenes have only one carbon atom between the aromatic rings, the presence of extra linkers between the aromatic rings allows the tuning of the desired cavity size, symmetry, and conformational mobility of the molecule. All-homocalixarenes have been defined by Vcgtle and co-workers as those in which all aromatic rings are separated by two carbon atoms. All-bis(homocalixarene)s are those separated by three carbon atoms, All-tris(homocalixarene)s separated by four carbon atoms, etc. We use the term homocalixarenes to refer to all calixarenes which have tether lengths greater than one carbon atom for at least one of the tethers and all-homocalixarenes for all calixarenes in which each of the tethers is greater than one carbon atom. The most direct method for the synthesis of all-homocalixarenes is the M ller–Rcscheisen cyclization which gives mixtures of calixarenes in low yields. All-homocalixarenes have also been made in a multistep synthesis by sulfur extrusion upon thermolysis of the corresponding sulfone. Several methods have been used in the synthesis of all-bis(homocalixarene)s, including alkylation of bis(bromomethyl) benzenes with tosylmethyl isocyanide and diethyl malonate, a Claisen rearrangement, and aryllithium/alkyl halide coupling. Larger all-homocalixarenes (n 4) have been made by the reaction of a bis(benzyllithium) with a,w-dihalides, and by the trimerization of a Fischer carbene complex. Homocalixarenes with heteroatoms in the linkers are much more widely studied compared to those with all-carbon linkers. The most popular of these are the bis(homooxacalix[3]arene)s which have enjoyed significant attention from researchers only after the discovery of a direct method for their preparation. Earlier reports from our laboratory have shown that the benzannulation reaction of Fischer carbene complexes can be used to synthesize paracyclophanes with various tether lengths. This chemistry was extended to the synthesis of calix[4]arenes, having various substituents, in a highly regiocontrolled triple annulation, and later extended to chiral calix[4]arenes. The purpose of the present work is to test the viability of this method to serve as a very general approach to all-homocalixarenes, having tethers with any number of desired carbon atoms, on a gram scale. One can envisage that both the homocalix[4]arenes 3 and homocalix[3]arenes 5 can be prepared by the triple annulation method (Scheme 1) involving the formation of two

Synthesis of [m.n]Cyclophanes: Regiochemistry Transfer from Vinyl Halides to Cyclophanes via Fischer Carbene Complexes

The double benzannulation of bis-carbene complexes of chromium with α,ω-diynes generates [m.n]cyclophanes in which all three rings are generated in a single reaction. This triple annulation process is very flexible allowing for the construction of symmetrical [n.n]cyclophanes and unsymmetrical [m.n]cyclophanes as well as isomers in which the two benzene rings are both meta bridged or both para bridged, and isomers that contain both meta and para bridges. The connectivity patterns of the bridges in the cyclophanes can be controlled by regioselectivity transfer from the bis-vinyl carbene complexes in which the substitution pattern of the vinyl groups in the carbene complexes dictate the connectivity pattern in the [m.n]cyclophanes. This synthesis of [n.n]cyclophanes is quite flexible with regard to ring size and can be used with tether lengths ranging from n=2 to n=16 and thus to ring sizes with up to 40 member rings. The only limitation to regioselectivity transfer from the carbene complexes to the [m.n]cyclophanes was found in the synthesis of para,para-cyclophanes with four carbon tethers for which the loss of fidelity occurred with the unexpected formation of meta,para-cyclophanes.

Long-Range Signaling in MutS and MSH Homologs via Switching of Dynamic Communication Pathways

Allostery is conformation regulation by propagating a signal from one site to another distal site. This study focuses on the long-range communication in DNA mismatch repair proteins MutS and its homologs where intramolecular signaling has to travel over 70 Å to couple lesion detection to ATPase activity and eventual downstream repair. Using dynamic network analysis based on extensive molecular dynamics simulations, multiple preserved communication pathways were identified that would allow such long-range signaling. The pathways appear to depend on the nucleotides bound to the ATPase domain as well as the type of DNA substrate consistent with previously proposed functional cycles of mismatch recognition and repair initiation by MutS and homologs. A mechanism is proposed where pathways are switched without major conformational rearrangements allowing for efficient long-range signaling and allostery.

10 How is fidelity maintained in nucleic acids? Two tales in DNA repair and DNA transcription from computer simulations

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