Simultaneous Acquisition of 15N- and 13C-Edited NOE Spectra of Proteins Dissolved in H2O

Pascal, Steven M.; Muhandiram, D. R.; Yamazaki, Toshio; Forman‐Kay, Julie D.; Kay, Lewis E.

doi:10.1006/jmrb.1994.1031

Cited by 249 publications

(142 citation statements)

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“…Aromatic resonances were assigned by using 2D hbCBcgcdHD or hbCBcgcdHDHE experiments (12). Proton-proton distance constraints were derived from 3D 13 (13,14). Restraints for torsion angles were derived from 3 J HNH ␣ measured from a 3D HNHA-J spectrum (15).…”

mentioning

confidence: 99%

Solution structure of dengue virus capsid protein reveals another fold

Jones

Groesch

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

312

421

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Dengue virus is responsible for Ϸ50 -100 million infections, resulting in nearly 24,000 deaths annually. The capsid (C) protein of dengue virus is essential for specific encapsidation of the RNA genome, but little structural information on the C protein is available. We report the solution structure of the 200-residue homodimer of dengue 2 C protein. The structure provides, to our knowledge, the first 3D picture of a flavivirus C protein and identifies a fold that includes a large dimerization surface contributed by two pairs of helices, one of which has characteristics of a coiled-coil. NMR structure determination involved a secondary structure sorting approach to facilitate assignment of the intersubunit nuclear Overhauser effect interactions. The dimer of dengue C protein has an unusually high net charge, and the structure reveals an asymmetric distribution of basic residues over the surface of the protein. Nearly half of the basic residues lie along one face of the dimer. In contrast, the conserved hydrophobic region forms an extensive apolar surface at a dimer interface on the opposite side of the molecule. We propose a model for the interaction of dengue C protein with RNA and the viral membrane that is based on the asymmetric charge distribution of the protein and is consistent with previously reported results.D engue virus, a member of the flavivirus genus of enveloped RNA viruses, is one of the most significant mosquito-borne viral pathogens, given the impact of the recent resurgence of dengue fever and dengue hemorrhagic fever (1). Other members of the flavivirus genus are also important human pathogens and include such viruses as yellow fever, West Nile virus, and Japanese encephalitis (2). The structure of dengue virus was recently described by using cryo-electron microscopy, which permitted visualization of certain viral protein components (3). The organization of the major envelope glycoprotein (E) in the virion was obtained by fitting the atomic structure of the ectodomain from the related tick-borne encephalitis (TBE) E protein into the outermost layer of density (4). Density internal to the E ectodomain demonstrated a host-derived lipid bilayer with transmembrane helices from E and the small structural protein M (5). Interior to the bilayer is the nucleocapsid core that comprises multiple copies of the capsid, or core, protein (C) and a single 10.7-kb genome RNA. Surprisingly, the organization of the C protein within the nucleocapsid core layer is not discernable. The lack of strong density for C may suggest a rather unique architecture of the flavivirus nucleocapsid core that is distinct from the structural organization of morphologically related viruses such as alphaviruses, a genus in the Togaviridae family of mosquito-borne viruses.The dengue C protein is essential in virus assembly to ensure specific encapsidation of the viral genome. The critical role of C is evident from the existence of subviral particles that are released from infected cells but lack C protein and genome RNA (6). The me...

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mentioning

confidence: 99%

Solution structure of dengue virus capsid protein reveals another fold

Jones

Groesch

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

312

421

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“…Backbone and side-chain resonance assignments were obtained using a set of gradient and sensitivity enhanced triple resonance experiment(HNCACB, CBCACONNH, CCC-TOCSY, HCC-TOCSY, HNCO, and HACACONNH) [76,77], supplemented with a simultaneous 15 N-, 13 C-edited NOESY-HSQC data set [78]. All experiments were performed at 25°C on a 500 MHz spectrometer.…”

Section: Nmr Spectroscopy 221 Resonance Assignmentsmentioning

confidence: 99%

“…Inter-proton distance restraints were obtained from a simultaneous 15 N-and 13 C-edited NOESY-HSQC data set [78] recorded with a mixing time of 150 ms (500 MHz). As the spectrum of I′ is characterized by poor chemical shift resolution, in particular for the side-chains, critical longrange methyl NOE restraints, including some involved in key non-native interactions, were obtained using higher resolution experiments, at a higher magnetic field.…”

Section: Noesy Distance Restraintsmentioning

confidence: 99%

Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

et al. 2011

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We have recently reported the atomic resolution structure of a low populated and transiently formed on-pathway folding intermediate of the FF domain from human HYPA/FBP11 [Korzhnev, D. M.; Religa, T. L.; Banachewicz, W.; Fersht, A. R.; Kay, L.E. Science 2011, 329, 1312–1316]. The structure was determined on the basis of backbone chemical shift and bond vector orientation restraints of the invisible intermediate state measured using relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy that were subsequently input into the database structure determination program, CS-Rosetta. As a cross-validation of the structure so produced, we present here the solution structure of a mimic of the folding intermediate that is highly populated in solution, obtained from the wild-type domain by mutagenesis that destabilizes the native state. The relaxation dispersion/CS-Rosetta structures of the intermediate are within 2 Å of those of the mimic, with the nonnative interactions in the intermediate also observed in the mimic. This strongly confirms the structure of the FF domain folding intermediate, in particular, and validates the use of relaxation dispersion derived restraints in structural studies of invisible excited states, in general.

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“…The NMR buffer also contained 10 % D 2 O for the deuterium lock. 15 N-labeled Pol λ BRCT was used to acquire the 3D 15 N-separated Nuclear Overhauser Enhancement Spectroscopy (NOESY) [16], 15 [17], Carr-Purcell Meiboom-Gill (CPMG) [18,19], and Residual Dipolar Coupling (RDC) experiments [20] with and without PF1 phage (Asla Biotech) [21]. The 13 C, 15 N-labeled Pol λ BRCT was used for all other experiments, including the 3D 13 C-separated NOESY [16].…”

Section: Expression and Purification Of The Human Pol λ Brct Domainmentioning

confidence: 99%

A comparison of BRCT domains involved in nonhomologous end-joining: Introducing the solution structure of the BRCT domain of polymerase lambda

et al. 2008

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Three of the four family X polymerases, DNA polymerase λ, DNA polymerase µ, and TdT have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase λ, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase λ BRCT domain reveals structural similarity to the BRCT domains of polymerase µ and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol λ plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol µ, Arg43. In contrast, the hydrophobic Leu60 residue of Pol λ contributes less significantly to binding than the structurally homologous Phe46 residue of Pol µ. A third leucine residue involved in the binding and activity of Pol µ, is nonconservatively replaced by a glutamine in Pol λ (Gln64) and, based on binding and activity data, is apparently unimportant for Pol λ interactions with the NHEJ complex. In conclusion, both the structure of the Pol λ BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol µ and TdT.

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Simultaneous Acquisition of 15N- and 13C-Edited NOE Spectra of Proteins Dissolved in H2O

Cited by 249 publications

References 0 publications

Solution structure of dengue virus capsid protein reveals another fold

Solution structure of dengue virus capsid protein reveals another fold

Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

A comparison of BRCT domains involved in nonhomologous end-joining: Introducing the solution structure of the BRCT domain of polymerase lambda

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