The CCPN data model for NMR spectroscopy: Development of a software pipeline

Vranken, Wim; Boucher, Wayne; Stevens, Tim J.; Fogh, Rasmus H.; Pajon, Anne; Llinás, Miguel; Ulrich, Eldon L.; Markley, John L.; Ionides, John; Laue, Ernest D.

doi:10.1002/prot.20449

Cited by 3,014 publications

(2,953 citation statements)

References 23 publications

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“…Data were processed with NMRPipe/NMRDRAW and analysed with CCPN software (Delaglio et al , 1995; Vranken et al , 2005). 15 N relaxation measurements and 1 H‐ 15 N heteronuclear NOE were collected as previously described (Kay et al , 1989).…”

Section: Methodsmentioning

confidence: 99%

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

et al. 2016

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TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.

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Section: Methodsmentioning

confidence: 99%

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

et al. 2016

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“…51 For the 15 Spectra were processed using NMRPipe, 52 and chemical shift assignments were made using the CCPNMR analysis software version 2.1. 53 Chemical shifts of rHPLC6 1 H and 15 N atoms were obtained from previously published studies on the synthetic protein, 28,54 and 15 N-TOCY and 15 N-NOESY experiments were used to assign resonances that underwent large chemical shifts in the rHPLC6-Arg 37 and rHPLC6-Ala 37 mutants. The R 1 and R 2 relaxation times were analyzed by fitting the cross-peak intensity decay to a two parameter, single exponential decay function I(t) ¼ I 0 e ÀtR1,2 , where I 0 is the intensity at time t ¼ 0 and I(t) is the intensity after a delay time t for the R 1 or R 2 experiment.…”

Section: Circular Dichroism Spectroscopymentioning

confidence: 99%

Increased flexibility decreases antifreeze protein activity

Patel

Graether

2010

Protein Science

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Antifreeze proteins protect several cold-blooded organisms from subzero environments by preventing death from freezing. The Type I antifreeze protein (AFP) isoform from Pseudopleuronectes americanus, named HPLC6, is a 37-residue protein that is a single a-helix. Mutational analysis of the protein showed that its alanine-rich face is important for binding to and inhibiting the growth of macromolecular ice. Almost all structural studies of HPLC6 involve the use of chemically synthesized protein as it requires a native N-terminal aspartate and an amidated C-terminus for full activity. Here, we examine the role of C-terminal amide and C-terminal arginine side chain in the activity, structure, and dynamics of nonamidated Arg 37 HPLC6, nonamidated HPLC6 Ala 37 , amidated HPLC6 Ala 37 , and fully native HPLC6 using a recombinant bacterial system. The thermal hysteresis (TH) activities of the nonamidated mutants are 35% lower compared with amidated proteins, but analysis of the NMR data and circular dichroism spectra shows that they are all still a-helical. Relaxation data from the two nonamidated mutants indicate that the C-terminal residues are considerably more flexible than the rest of the protein because of the loss of the amide group, whereas the amidated Ala 37 mutant has a C-terminus that is as rigid as the wild-type protein and has high TH activity. We propose that an increase in flexibility of the AFP causes it to lose activity because its dynamic nature prevents it from binding strongly to the ice surface.

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“…The constant time period was set to 26 ms. NMR experiments were recorded on a Bruker DMX or Avance III 600 MHz NMR spectrometer, equipped with a TCI-Z-GRAD cryoprobe at 298 K. NMR data were processed using nmrPipe 35 and analyzed using CCPN Analysis 2.1.5. 36 The Src SH3 domain resonances were assigned with the help of assignments reported for the chicken Src SH3−SH2 domains. 37 The transverse relaxation rate of resonances in the diamagnetic sample (R dia H ) was determined after processing with a 2 Hz line-broadening exponential window function.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

A Focal Adhesion Kinase-Derived Peptide Binds the Src SH3 Domain in Two Orientations, As Demonstrated Using Paramagnetic Nuclear Magnetic Resonance

et al. 2015

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SH3 binding peptides contain polyproline helices and are classified according to their binding orientations as N-to-C-terminal or C-to-N-terminal. We have tested the hypothesis that such a peptide binds in both orientations but with different populations. A focal adhesion kinase (FAK)-derived peptide was tested for its binding orientation on the Src SH3 domain. Paramagnetic tags were introduced at several positions on the SH3 domain, and on the basis of the paramagnetic relaxation enhancements (PREs) of the amide protons, the positions of the paramagnetic centers were determined. Two peptides were synthesized with 13 C-enriched Ala or Pro, at the N-terminal or C-terminal side of the peptide, and the intermolecular PREs were measured. The results provide compelling evidence that the FAK-derived peptide binds the SH3 domain in two orientations. In the major state, the SH3 domain binds the peptide in the N−C orientation, whereas 20% of the time, the peptide binds in the C−N orientation. We conclude that the distinction between N− C and C−N orientations, which is based on crystal structures, might be artificial. The pseudosymmetric nature of the polyproline helix might allow for binding in both orientations in the solution state.

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The CCPN data model for NMR spectroscopy: Development of a software pipeline

Cited by 3,014 publications

References 23 publications

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Increased flexibility decreases antifreeze protein activity

A Focal Adhesion Kinase-Derived Peptide Binds the Src SH3 Domain in Two Orientations, As Demonstrated Using Paramagnetic Nuclear Magnetic Resonance

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