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- Carlström, Göran, et al.
(författare)
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[7] NMR studies of complex DNA structures : The holliday junction intermediate in genetic recombination
- 1995
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Ingår i: Methods in Enzymology : Nuclear Magnetic Resonance and Nucleic Acids - Nuclear Magnetic Resonance and Nucleic Acids. - 0076-6879. - 9780121821623 ; 261, s. 163-182
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Bokkapitel (refereegranskat)abstract
- Publisher Summary This chapter discusses the current status, of using nuclear magnetic resonance (NMR), to study the structure and dynamics of the holliday junction (HJ). Complex deoxyribonucleic acid (DNA) structures (e.g., triplexes, quadruplexes, junctions) pose difficult problems for study, by NMR, relative to the typical DNA duplexes, because they have nonstandard or distorted local conformations and higher molecular weights that give rise to large resonance linewidths and severe 1H spectral overlap. With more atoms in the system, both assignment and structure calculation become more challenging. The HJ, a four-arm DNA crossover structure, is a transient intermediate formed in the course of genetic recombination as well as during other cellular processes, such as replication and telomere resolution. A significant body of evidence has accumulated, indicating that the structure at the junction has a central role, in determining the outcome of these cellular events. For NMR studies, the titration of the four component 16-mer strands to create an equimolar mixture is critical. Gel electrophoresis has shown that titrations based on the standard ultraviolet (UV) estimates of strand concentrations result in significant amounts of residual single-strand, half-complementary duplex, and three-arm structures.
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| 2. |
- Potts, Barbara C.M., et al.
(författare)
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1H NMR assignments of apo calcyclin and comparative structural analysis with calbindin d(9k) and s 100β
- 1996
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 5:11, s. 2162-2174
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Tidskriftsartikel (refereegranskat)abstract
- The homodimeric S100 protein calcyclin has been studied in the apo state by two-dimensional 1H NMR spectroscopy. Using a combination of scalar correlation and NOE experiments, sequence-specific 1H NMR assignments were obtained for all but one backbone and >90% of the side-chain resonances. To our knowledge, the 2 x 90 residue (20 kDa) calcyclin dimer is the largest protein system for which such complete assignments have been made by purely homonuclear methods. Sequential and medium-range NOEs and slowly exchanging backbone amide protons identified directly the four helices and the short antiparallel β-type interaction between the two binding loops that comprise each subunit of the dimer. Further analysis of NOEs enabled the unambiguous assignment of 556 intrasubunit distance constraints, 24 intrasubunit hydrogen bonding constraints, and 2 x 26 intersubunit distance constraints. The conformation of the monomer subunit was refined by distance geometry and restrained molecular dynamics calculations using the intrasubunit constraints only. Calculation of the dimer structure starting from this conformational ensemble has been reported elsewhere. The extent of structural homology among the apo calcyclin subunit, the monomer subunit of apo S100β, and monomeric apo calbindin D(9k) has been examined in detail by comparing 1H NMR chemical shifts and secondary structures. This analysis was extended to a comprehensive comparison of the three-dimensional structures of the calcyclin monomer subunit and calbindin D(9k), which revealed greater similarity in the packing of their hydrophobic cores than was anticipated previously. Together, these results support the hypothesis that all members of the S100 family have similar core structures and similar modes of dimerization. Analysis of the amphiphilicity of Helix IV is used to explain why calbindin D(9k) is monomeric, but full-length S100 proteins form homodimers.
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