Alexey K. Shaytan

Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses

Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.

Physicochemical mechanisms of protein regulation by phosphorylation

Phosphorylation offers a dynamic way to regulate protein activity and subcellular localization, which is achieved through its reversibility and fast kinetics. Adding or removing a dianionic phosphate group somewhere on a protein often changes the protein’s structural properties, its stability and dynamics. Moreover, the majority of signaling pathways involve an extensive set of protein–protein interactions, and phosphorylation can be used to regulate and modulate protein–protein binding. Losses of phosphorylation sites, as a result of disease mutations, might disrupt protein binding and deregulate signal transduction. In this paper we focus on the effects of phosphorylation on protein stability, dynamics, and binding. We describe several physico-chemical mechanisms of protein regulation through phosphorylation and pay particular attention to phosphorylation in protein complexes and phosphorylation in the context of disorder–order and order–disorder transitions. Finally we assess the role of multiple phosphorylation sites in a protein molecule, their possible cooperativity and function.

Self-assembling nanofibers from thiophene-peptide diblock oligomers: a combined experimental and computer simulations study.

We report herein the synthesis of a novel type of hybrid compound that consists of a poly(ethylene oxide) (PEO) functionalized β-sheet peptide sequence covalently linked to an alkylated quaterthiophene moiety. Compounds of this class are highly promising for technological applications because their self-assembly and stimuli-responsive behavior, which is mainly caused by the peptide moieties, combined with the potential semiconducting properties of oligothiophenes provides unprecedented opportunities for the design of advanced materials at the nanoscale in such areas as, for example, organic electronics and sensor design for chemical and biomedical applications. The compound presented herein is experimentally shown to form stable fibrillar aggregates that are visualized by both transmission electron and atomic force microscopy. We developed a theoretical methodology to study the possible intermolecular arrangements and their characteristic features with the help of all-atom MD simulations, while simultaneously incorporating available experimental data into the model. Large-scale atomistic simulations of several fibrillar aggregates with different molecular arrangements were performed. The results of the simulations are compared with experimental data, which leads to the proposition of a likely model for the arrangement of the individual molecules within the observed aggregates.

Nucleosome adaptability conferred by sequence and structural variations in histone H2A-H2B dimers.

Nucleosome variability is essential for their functions in compacting the chromatin structure and regulation of transcription, replication and cell reprogramming. The DNA molecule in nucleosomes is wrapped around an octamer composed of four types of core histones (H3, H4, H2A, H2B). Nucleosomes represent dynamic entities and may change their conformation, stability and binding properties by employing different sets of histone variants or by becoming post-translationally modified. There are many variants of histones H2A and H2B. Specific H2A and H2B variants may preferentially associate with each other resulting in different combinations of variants and leading to the increased combinatorial complexity of nucleosomes. In addition, the H2A-H2B dimer can be recognized and substituted by chaperones/remodelers as a distinct unit, can assemble independently and is stable during nucleosome unwinding. In this review we discuss how sequence and structural variations in H2A-H2B dimers may provide necessary complexity and confer the nucleosome functional variability.

Structural analysis of nucleosomal barrier to transcription

Significance On the majority of eukaryotic genes RNA polymerase II meets nucleosomes during transcription of every ∼200 bp of DNA. The key features of Pol II–nucleosome encounter are conserved from yeast to human, but the molecular mechanism of this process remains unknown. Our data suggest a mechanism of formation of the high nucleosomal barrier to Pol II that participates in regulation of transcript elongation in eukaryotes. The proposed mechanism explains the remarkable efficiency of nucleosome survival during transcription, important for maintenance of epigenetic and regulatory histone modifications. Similar mechanisms are likely used during various other DNA transactions, including DNA replication and ATP-dependent chromatin remodeling. Some factors involved in chromatin transcription (e.g., FACT and PARP) participate in cancer development/aging. Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. Here we show that both DNA–histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone–histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA–protein and protein–protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed.

Analysis of the mechanism of nucleosome survival during transcription

Maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. Our previous data identified a key intermediate (a small intranucleosomal DNA loop, Ø-loop) that is likely required for nucleosome survival during transcription by RNA polymerase II (Pol II) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. To evaluate these predictions, we analysed transcription through a nucleosome by different, structurally related RNA polymerases and mutant yeast Pol II having different histone-interacting surfaces that presumably stabilize the Ø-loop. The height of the nucleosomal barrier to transcription and efficiency of nucleosome survival correlate with the net negative charges of the histone-interacting surfaces. Molecular modeling and analysis of Pol II-nucleosome intermediates by DNase I footprinting suggest that efficient Ø-loop formation and nucleosome survival are mediated by electrostatic interactions between the largest subunit of Pol II and core histones.

Voltage-gated ion channel modulation by lipids: Insights from molecular dynamics simulations

Cells commonly use lipids to modulate the function of ion channels. The lipid content influences the amplitude of the ionic current and changes the probability of voltage-gated ion channels being in the active or in the resting states. Experimental findings inferred from a variety of techniques and molecular dynamics studies have revealed a direct interaction between the lipid headgroups and the ion channel residues, suggesting an influence on the ion channel function. On the other hand the alteration of the lipids may in principle modify the overall electrostatic environment of the channel, and hence the transmembrane potential, leading to an indirect modulation, i.e. a global effect. Here we have investigated the structural and dynamical properties of the voltage-gated potassium channel Kv1.2 embedded in bilayers with modified upper or lower leaflet compositions corresponding to realistic biological scenarios: the first relates to the effects of sphingomyelinase, an enzyme that modifies the composition of lipids of the outer membrane leaflets, and the second to the effect of the presence of a small fraction of PIP2, a highly negatively charged lipid known to modulate voltage-gated channel function. Our molecular dynamics simulations do not enable to exclude the global effect mechanism in the former case. For the latter, however, it is shown that local interactions between the ion channel and the lipid headgroups are key-elements of the modulation.

HistoneDB 2.0: a histone database with variants—an integrated resource to explore histones and their variants

Compaction of DNA into chromatin is a characteristic feature of eukaryotic organisms. The core (H2A, H2B, H3, H4) and linker (H1) histone proteins are responsible for this compaction through the formation of nucleosomes and higher order chromatin aggregates. Moreover, histones are intricately involved in chromatin functioning and provide a means for genome dynamic regulation through specific histone variants and histone post-translational modifications. ‘HistoneDB 2.0 – with variants’ is a comprehensive database of histone protein sequences, classified by histone types and variants. All entries in the database are supplemented by rich sequence and structural annotations with many interactive tools to explore and compare sequences of different variants from various organisms. The core of the database is a manually curated set of histone sequences grouped into 30 different variant subsets with variant-specific annotations. The curated set is supplemented by an automatically extracted set of histone sequences from the non-redundant protein database using algorithms trained on the curated set. The interactive web site supports various searching strategies in both datasets: browsing of phylogenetic trees; on-demand generation of multiple sequence alignments with feature annotations; classification of histone-like sequences and browsing of the taxonomic diversity for every histone variant. HistoneDB 2.0 is a resource for the interactive comparative analysis of histone protein sequences and their implications for chromatin function. Database URL: http://www.ncbi.nlm.nih.gov/projects/HistoneDB2.0

Solvent Accessible Surface Area of Amino Acid Residues in Globular Proteins: Correlation of Apparent Transfer Free Energies with Experimental Hydrophobicity Scales

It is known that the distribution of amino acid residues in globular proteins between surface and interior is in certain correlation with various experimental scales based on partitioning of amino acids or their analogs between water and organic solvents. These scales are often used in various quantitative structure-activity relationship (QSAR) studies as well as for evaluation of stability of proteins. In this work we have analyzed the distribution of residues based on their solvent accessible surface area in more than 8000 protein structures. Using extensive statistical sampling, we have computed residue apparent free energies of transfer between protein interior and surface applying various criteria for classifying residues as exposed or buried. The correlation of these statistical energies with several experimental hydrophobicity scales is discussed. We propose three types of statistical apparent transfer free energy scales and show that each of these scales is in better correlation with one of the experimental hydrophobicity scales (water/vapor, water/cyclohexane, and water/octanol transfer scales). The data are interpreted through the application of theoretical considerations by Finkelstein et al. (Protein Struct. Funct. Genet. 1995, 23, 142) based on random energy model of heteropolymer globules. The deviation of apparent transfer free energies from experimental scales is discussed and analyzed. The variations of amino acid distribution in proteins with the size of protein structure is discussed and the final protein set is chosen to minimize these variations.

Comparative computational study of interaction of C60-fullerene and tris-malonyl-C60-fullerene isomers with lipid bilayer: relation to their antioxidant effect.

Oxidative stress induced by excessive production of reactive oxygen species (ROS) has been implicated in the etiology of many human diseases. It has been reported that fullerenes and some of their derivatives–carboxyfullerenes–exhibits a strong free radical scavenging capacity. The permeation of C60-fullerene and its amphiphilic derivatives–C3-tris-malonic-C60-fullerene (C3) and D3-tris-malonyl-C60-fullerene (D3)–through a lipid bilayer mimicking the eukaryotic cell membrane was studied using molecular dynamics (MD) simulations. The free energy profiles along the normal to the bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) for C60, C3 and D3 were calculated. We found that C60 molecules alone or in clusters spontaneously translocate to the hydrophobic core of the membrane and stay inside the bilayer during the whole period of simulation time. The incorporation of cluster of fullerenes inside the bilayer changes properties of the bilayer and leads to its deformation. In simulations of the tris-malonic fullerenes we discovered that both isomers, C3 and D3, adsorb at the surface of the bilayer but only C3 tends to be buried in the area of the lipid headgroups forming hydrophobic contacts with the lipid tails. We hypothesize that such position has implications for ROS scavenging mechanism in the specific cell compartments.