На главную              К списку выставок

Современные достижения в области биохимических исследований

Журнальные статьи

1.U17232
Battistini F. et al. Structure-Based Identification of New High-Affinity Nucleosome Binding Sequences // J. Mol. Biol. 2012. Vol. 420, № 1-2. P. 8–16.

The substrate for the proteins that express genetic information in the cell is not naked DNA but an assembly of nucleosomes, where the DNA is wrapped around histone proteins. The organization of these nucleosomes on genomic DNA is influenced by the DNA sequence. Here, we present a structure-based computational approach that translates sequence information into the energy required to bend DNA into a nucleosome-bound conformation. The calculations establish the relationship between DNA sequence and histone octamer binding affinity. In silico selection using this model identified several new DNA sequences, which were experimentally found to have histone octamer affinities comparable to the highest-affinity sequences known. The results provide insights into the molecular mechanism through which DNA sequence information encodes its organization. A quantitative appreciation of the thermodynamics of nucleosome positioning and rearrangement will be one of the key factors in understanding the regulation of transcription and in the design of new promoter architectures for the purposes of tuning gene expression dynamics. (C) 2012 Elsevier Ltd. All rights reserved.

2.U04533
Cui P. et al. A novel mechanism of epigenetic regulation: Nucleosome-space occupancy // Biochem. Biophys. Res. Commun. 2010. Vol. 391, № 1. P. 884–889.

Nucleosome positioning around the gene space (or transcriptional unit) plays a crucial role for gene regulation but we do not know if the spatial organization-nucleosome-space occupancy or nucleosome density in a defined sequence unit length-contributes to the regulation complexity of mammalian gene expression. Using our own rmRNA-Seq (ribosomal RNA-minus RNA sequencing) and publically available ChIP-Seq (H3) data from mouse stem cells. we discovered a non-random distribution of nucleosomes along chromosomes, and further genome-wide studies oil histone modifications, DNA methylation, transcriptional activity, gene density, and base compositional dynamics, demonstrated that nucleosome-space Occupancy of genomic regions-clustered genes and their intergenic spaces-show distinctive features, where a high occupancy coincides with active transcription, intensive histone modifications, poor DNA methylation, and higher GC contents as compared to the nucleosome-poor regions. We therefore proposed that nucleosome-space Occupancy as a novel mechanism of epigenetic gene regulation, creating a vital environment for transcriptional activation. (C) 2009 Elsevier Inc. All rights reserved.

3.U15453
Gu X., Schroeder S.J. Different Sequences Show Similar Quaternary Interaction Stabilities in Prohead Viral RNA Self-assembly // J. Biol. Chem. 2011. Vol. 286, № 16. P. 14419–14426.

Prohead RNA (pRNA) is an essential component of the self-assembling phi 29 bacteriophage DNA packaging motor. Different related species of bacteriophage share only 12% similarity in pRNA sequences. The secondary structure for pRNA is conserved, however. In this study, we present evidence for self-assembly in different pRNA sequences and new measurements of the energetics for the quaternary interactions in pRNA dimers and trimers. The energetics for self-assembly in different pRNA sequences are similar despite very different sequences in the loop-loop interactions. The architecture surrounding the interlocking loops contributes to the stability of the pRNA quaternary interactions, and sequence variation outside the interlocking loops may counterbalance the changes in the loop sequences. Thus, the evolutionary divergence of pRNA sequences maintains not only conservation of function and secondary structure but also stabilities of quaternary interactions. The self-assembly of pRNA can be fine-tuned with variations in magnesium chloride, sodium chloride, temperature, and concentration. The ability to control pRNA self-assembly holds promise for the development of nanoparticle therapeutic applications for this biological molecule. The pRNA system is well suited for future studies to further understand the energetics of RNA tertiary and quaternary interactions, which can provide insight into larger biological assemblies such as viruses and biomolecular motors.

4.U01982
Hecht A.H. et al. Aptamers as Affinity Reagents in an Integrated Electrophoretic Lab-on-a-Chip Platform // Anal. Chem. 2010. Vol. 82, № 21. P. 8813–8820.

Nucleic acid based affinity reagents (e.g., aptamers) offer several possible advantages over antibodies as specific recognition elements in biochemical assays. Besides offering improved cost and stability, aptamers are ideal for rapid electrophoretic analysis due to their low molecular weight and high negative charge. While aptamers have proven well-suited for affinity-shift electrophoretic analysis, demonstrating a fully integrated aptamer-based assay platform represents an important achievement toward low-cost point-of-care analysis, particularly for remote or resource poor settings where cost and ambient stability of reagents is a key consideration. Here we perform and evaluate the suitability of aptamer-based affinity assays for two clinically relevant target analytes (IgE using a known aptamer and NF-kappa B using a thiomodified aptamer) in an integrated electrophoretic gel-shift platform. Key steps of (i) mixing sample with aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated on-chip upstream of a fluorescence-based gel-shift analysis step. This approach, utilizing a size-exclusion membrane optimized here for aptamer retention and preconcentration with sample, enables automated sample-to-answer for trace analytes in 10 min or less. We addressed notable nonspecific interference from serum proteins by adding similar nucleic acid competitors to suppress such interactions with the aptamer. Nanomolar sensitivities were demonstrated and integrated preconcentration of sample provides an important means of further improving detection sensitivities. Aptamers proved superior in many respects to antibody reagents, particularly with regard to speed and resolution of gel-shifts associated with specific binding to target

5.U55713
Henriksen N.M., Davis D.R., Cheatham T.E. Molecular dynamics re-refinement of two different small RNA loop structures using the original NMR data suggest a common structure // J. Biomol. NMR. 2012. Vol. 53, № 4. P. 321–339.

Restrained molecular dynamics simulations are a robust, though perhaps underused, tool for the end-stage refinement of biomolecular structures. We demonstrate their utility-using modern simulation protocols, optimized force fields, and inclusion of explicit solvent and mobile counterions-by re-investigating the solution structures of two RNA hairpins that had previously been refined using conventional techniques. The structures, both domain 5 group II intron ribozymes from yeast ai5 gamma and Pylaiella littoralis, share a nearly identical primary sequence yet the published 3D structures appear quite different. Relatively long restrained MD simulations using the original NMR restraint data identified the presence of a small set of violated distance restraints in one structure and a possibly incorrect trapped bulge nucleotide conformation in the other structure. The removal of problematic distance restraints and the addition of a heating step yielded representative ensembles with very similar 3D structures and much lower pairwise RMSD values. Analysis of ion density during the restrained simulations helped to explain chemical shift perturbation data published previously. These results suggest that restrained MD simulations, with proper caution, can be used to "update" older structures or aid in the refinement of new structures that lack sufficient experimental data to produce a high quality result. Notable cautions include the need for sufficient sampling, awareness of potential force field bias (such as small angle deviations with the current AMBER force fields), and a proper balance between the various restraint weights.Times Cited: 7 (from All Databases)

6.U15453
Jagannathan I., Pepenella S., Hayes J.J. Activity of FEN1 Endonuclease on Nucleosome Substrates Is Dependent upon DNA Sequence but Not Flap Orientation // J. Biol. Chem. 2011. Vol. 286, № 20. P. 17521–17529.

AT-rich DNA is concentrated in the nucleosome-free regions (NFRs) associated with transcription start sites of most genes. We tested the hypothesis that AT-rich DNA engenders NFR formation by virtue of its rigidity and consequent exclusion of nucleosomes. We found that the AT-rich sequences present in many NFRs have little effect on the stability of nucleosomes. Rather, these sequences facilitate the removal of nucleosomes by the RSC chromatin remodeling complex. RSC activity is stimulated by AT-rich sequences in nucleosomes and inhibited by competition with AT-rich DNA. RSC may remove NFR nucleosomes without effect on adjacent ORF nucleosomes. Our findings suggest that many NFRs are formed and maintained by an active mechanism involving the ATP-dependent removal of nucleosomes rather than a passive mechanism due to the intrinsic instability of nucleosomes on AT-rich DNA sequences.

7.U49676
Krivega I., Dale R.K., Dean A. Role of LDB1 in the transition from chromatin looping to transcription activation // Genes Dev. 2014. Vol. 28, № 12. P. 1278–1290.

AT-rich DNA is concentrated in the nucleosome-free regions (NFRs) associated with transcription start sites of most genes. We tested the hypothesis that AT-rich DNA engenders NFR formation by virtue of its rigidity and consequent exclusion of nucleosomes. We found that the AT-rich sequences present in many NFRs have little effect on the stability of nucleosomes. Rather, these sequences facilitate the removal of nucleosomes by the RSC chromatin remodeling complex. RSC activity is stimulated by AT-rich sequences in nucleosomes and inhibited by competition with AT-rich DNA. RSC may remove NFR nucleosomes without effect on adjacent ORF nucleosomes. Our findings suggest that many NFRs are formed and maintained by an active mechanism involving the ATP-dependent removal of nucleosomes rather than a passive mechanism due to the intrinsic instability of nucleosomes on AT-rich DNA sequences.

8.U15561
Lawrimore J., Bloom K.S., Salmon E.D. Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome // J. Cell Biol. 2011. Vol. 195, № 4. P. 573–582.

Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of. similar to 5 Cse4s, similar to 3 inner kinetochore CBF3 complexes, and. similar to 20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.

9. U49676
Lorch Y., Maier-Davis B., Kornberg R.D. Role of DNA sequence in chromatin remodeling and the formation of nucleosome-free regions // Genes Dev. 2014. Vol. 28, № 22. P. 2492–2497.

AT-rich DNA is concentrated in the nucleosome-free regions (NFRs) associated with transcription start sites of most genes. We tested the hypothesis that AT-rich DNA engenders NFR formation by virtue of its rigidity and consequent exclusion of nucleosomes. We found that the AT-rich sequences present in many NFRs have little effect on the stability of nucleosomes. Rather, these sequences facilitate the removal of nucleosomes by the RSC chromatin remodeling complex. RSC activity is stimulated by AT-rich sequences in nucleosomes and inhibited by competition with AT-rich DNA. RSC may remove NFR nucleosomes without effect on adjacent ORF nucleosomes. Our findings suggest that many NFRs are formed and maintained by an active mechanism involving the ATP-dependent removal of nucleosomes rather than a passive mechanism due to the intrinsic instability of nucleosomes on AT-rich DNA sequences.

10.U15453
Patel A. et al. Identification of Residues in Chromodomain Helicase DNA-Binding Protein 1 (Chd1) Required for Coupling ATP Hydrolysis to Nucleosome Sliding // J. Biol. Chem. 2011. Vol. 286, № 51. P. 43984–43993.

CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which provide a structural explanation for the regulation of CHD4 activities by intramolecular domain communication. Our results demonstrate functional interdependency between domains within a chromatin remodeller. (c) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

11.U49676
Seeber A., Dion V., Gasser S.M. Checkpoint kinases and the INO80 nucleosome remodeling complex enhance global chromatin mobility in response to DNA damage // Genes Dev. 2013. Vol. 27, № 18. P. 1999–2008.

Lacking from the rapidly evolving field of chromatin regulation is a discrete model of chromatin states. We propose that each state in such a model should meet two conditions: a structural component and a quantifiable effect on transcription. The practical benefits to the field of a model with greater than two states (including one with six states, as described herein) would be to improve interpretation of data from disparate organ systems, to reflect temporal and developmental dynamics and to integrate the, at present, conceptually and experimentally disparate analyses of individual genetic loci (in vitro or using single gene approaches) and genome-wide features (including ChIPseq, chromosomal capture and mRNA expression via microarrays/sequencing). (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

12.U03386
Singh M.P., Wijeratne S.S.K., Zempleni J. Biotinylation of lysine 16 in histone H4 contributes toward nucleosome condensation // Arch. Biochem. Biophys. 2013. Vol. 529, № 2. P. 105–111.

Holocarboxylase synthetase (HLCS) is part of a multiprotein gene repression complex and catalyzes the covalent binding of biotin to lysines (K) in histones H3 and H4, thereby creating rare gene repression marks such as K16-biotinylated histone H4 (H4K16bio). We tested the hypothesis that H4K16bio contributes toward nucleosome condensation and gene repression by HLCS. We used recombinant histone H4 in which K16 was mutated to a cysteine (H4K16C) for subsequent chemical biotinylation of the sulfhydryl group to create H4K16Cbio. Nucleosomes were assembled by using H4K16Cbio and the 'Widom 601' nucleosomal DNA position sequence; biotin-free histone H4 and H4K16C were used as controls. Nucleosomal compaction was analyzed using atomic force microscopy (AFM). The length of DNA per nucleosome was similar to 30% greater in H4K16Cbio-containing histone octamers (61.14 +/- 10.92 nm) compared with native H4 (46.89 +/- 12.6 nm) and H4K16C (47.26 +/- 10.32 nm), suggesting biotin-dependent chromatin condensation (P < 0.001). Likewise, the number of DNA turns around histone core octamers was similar to 17.2% greater in in H4K16Cbio-containing octamers (1.78 +/- 0.16) compared with native H4 (1.52 +/- 0.21) and H4K16C (1.52 +/- 0.17), judged by the rotation angle (P < 0.001; N = 150). We conclude that biotinylation of K16 in histone H4 contributes toward chromatin condensation. (C) 2012 Elsevier Inc. All rights reserved.

13.U49676
Son J. et al. Nucleosome-binding activities within JARID2 and EZH1 regulate the function of PRC2 on chromatin // Genes Dev. 2013. Vol. 27, № 24. P. 2663–2677.

By systematic analysis of high-throughput sequencing datasets from the human genome, we found that protein-coding genes have a specific chromatin structure near transcription termination sites relative to non-coding genes, one related to polyadenylation. Nucleosome was depleted near the site of cleavage/polyadenylation (polyA site) regardless of its relative position in the gene. DNA sequence plays an improtant role in nucleosome distribution, and conservative sequence elements and the protein binding to them are major determinants in causing nucleosome depletion near polyA sites. Furthermore, nucleosome occupancy was regulated by gene transcription and RNA polymerase II (RNAPII) occupancy. Our results reveal influences on nucleosome occupancy near polyadenylation sites and constitute evidence indicating that nucleosome distribution regulates 3' end processing of protein-coding genes.

14.U17232
Stocks B.B. et al. Temporal Development of Protein Structure during S100A11 Folding and Dimerization Probed by Oxidative Labeling and Mass Spectrometry // J. Mol. Biol. 2011. Vol. 409, № 4. P. 669–679.

Considerable progress in deciphering the mechanisms of protein folding has been made. However, most work in this area has focused on single-chain systems, whereas the majority of proteins are oligomers. The spontaneous assembly of intact multi-subunit systems from disordered building blocks encompasses the formation of intramolecular as well as intermolecular contacts. Both types of interaction affect the solvent accessibility of individual protein segments. This work employs pulsed hydroxyl radical (center dot OH) labeling for tracking time-dependent accessibility changes during folding and assembly of the S100A11 homodimer. center dot OH induces covalent modifications at exposed residues. Structural snapshots are obtained by combining center dot OH labeling with rapid mixing and mass spectrometry. The free subunits are found to possess a partially non-native hydrophobic core that prevents subunit association during the initial stages of the reaction. Instead, the protein forms an early (10 ms) monomeric intermediate that exhibits reduced solvent accessibility in regions distant from helices I and IV, which constitute the dimerization interface. Subunit association is complete after 800 ms, although the protein retains significant disorder in helices II and III at this point. Subsequent consolidation of these elements leads to the native state. The experimental strategy used here could become a general tool for deciphering kinetic mechanisms of biomolecular self-assembly processes. (C) 2011 Elsevier Ltd. All rights reserved.

15.U57390
Strien J., Sanft J., Mall G. Enhancement of PCR Amplification of Moderate GC-Containing and Highly GC-Rich DNA Sequences // Mol. Biotechnol. 2013. Vol. 54, № 3. P. 1048–1054.

PCR is a commonly used and highly efficient technique in biomolecular laboratories for specific amplification of DNA. However, successful DNA amplification can be very time consuming and troublesome because many factors influence PCR efficiency. Especially GC-rich DNA complicates amplification because of generation of secondary structures that hinder denaturation and primer annealing. We investigated the impact of previously recommended additives such as dimethylsulfoxide (DMSO), magnesium chloride (MgCl2), bovine serum albumin (BSA), or formamide. Furthermore, we tested company-specific substances as Q-Solution, High GC Enhancer, and Hi-Spec; various actively promoted polymerases as well as different PCR conditions for their positive effects on DNA amplification of templates with moderate and extremely high CG-content. We found considerable differences of specificity and quantity of product between different terms. In this article, we introduce conditions for optimized PCR to help resolve problems amplifying moderate to high GC-rich templates.

16.U17232
Tims H.S. et al. Dynamics of Nucleosome Invasion by DNA Binding Proteins // J. Mol. Biol. 2011. Vol. 411, № 2. P. 430–448.

Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome. (C) 2011 Elsevier Ltd. All rights reserved.

17.U4536X
Tseng T.-S. et al. A molten globule-to-ordered structure transition of Drosophila melanogaster crammer is required for its ability to inhibit cathepsin // Biochem. J. 2012. Vol. 442. P. 563–572.

Drosophila melanogaster crammer is a novel cathepsin inhibitor that is involved in LTM (long-term memory) formation. The mechanism by which the inhibitory activity is regulated remains unclear. In the present paper we have shown that the oligomeric state of crammer is pH dependent. At neutral pH, crammer is predominantly dimeric in vitro as a result of disulfide bond formation, and is monomeric at acidic pH. Our inhibition assay shows that monomeric crammer, not disulfide-bonded dimer, is a strong competitive inhibitor of cathepsin L. Crammer is a monomeric molten globule in acidic solution, a condition that is similar to the environment in the lysosome where crammer is probably located. Upon binding to cathepsin L, however, crammer undergoes a molten globule-to-ordered structural transition. Using high-resolution NMR spectroscopy, we have shown that a cysteine-to-serine point mutation at position 72 (C72S) renders crammer monomeric at pH 6.0 and that the structure of the C72S variant highly resembles that of wild-type crammer in complex with cathepsin L at pH 4.0. We have determined the first solution structure of propeptide-like protease inhibitor in its active form and examined in detail using a variety of spectroscopic methods the folding properties of crammer in order to delineate its biomolecular recognition of cathepsin.

18.U17232
Yamashita H. et al. Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM // J. Mol. Biol. 2012. Vol. 422, № 2. P. 300–309.

Advances in microscopy have contributed to many biologic discoveries. Electron microscopic techniques such as cryo-electron tomography are remarkable tools for imaging the interiors of bacterial cells in the near-native state, whereas optical microscopic techniques such as fluorescence imaging are useful for following the dynamics of specific single molecules in living cells. Neither technique, however, can be used to visualize the structural dynamics of a single molecule at high resolution in living cells. In the present study, we used high-speed atomic force microscopy (HS-AFM) to image the molecular dynamics of living bacterial cell surfaces. HS-AFM visualizes the dynamic molecular processes of isolated proteins at sub-molecular resolution without the need for complicated sample preparation. In the present study, magnetotactic bacterial cells were anchored in liquid medium on substrate modified by poly-L-lysine and glutaraldehyde. High-resolution HS-AFM images of live cell surfaces showed that the bacterial outer membrane was covered with a net-like structure comprising holes and the hole rims framing them. Furthermore, HS-AFM captured the dynamic movement of the surface ultrastructure, showing that the holes in the net-like structure slowly diffused in the cell surface. Nano-dissection revealed that porin trimers constitute the net-like structure. Here, we report for the first time the direct observation of dynamic molecular architectures on a live cell surface using HS-AFM.

На главную              К списку выставок