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Bertini, Ivano; Donaire, Antonio; Jiménez, Beatriz; Luchinat, Claudio; Parigi, Giacomo; Piccioli, Mario; Poggi, Luisa

doi:10.1023/a:1012422402545

Cited by 110 publications

(117 citation statements)

References 28 publications

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“…Distance constraints were extracted from a 1 H homonuclear NOESY spectrum (mixing time, 150 ms) and 15 N and 13 C HSQC-NOESY spectra. angles were calculated on the basis of HNHA spectra and angles as described previously (23,24). Hydrogen bond donors were extracted from 15 N HSQC spectra recorded on protein samples in D 2 O.…”

Section: Methodsmentioning

confidence: 99%

Structural Analysis of the Mitotic Regulator hPin1 in Solution

Bayer

Goettsch

Mueller

et al. 2003

Journal of Biological Chemistry

118

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The peptidyl-prolyl cis/trans isomerase hPin1 is a phosphorylation-dependent regulatory enzyme whose substrates are proteins involved in regulation of cell cycle, transcription, Alzheimer's disease, and cancer pathogenesis. We have determined the solution structure of the two domain protein hPin1-(1-163) and its separately expressed PPIase domain (50 -163) (hPin1 PPIase ) with an root mean square deviation of <0.5 Å over backbone atoms using NMR. Domain organization of hPin1 differs from that observed in structures solved by x-ray crystallography. Whereas PPIase and WW domain are tightly packed onto each other and share a common binding interface in crystals, our NMR-based data revealed only weak interaction of both domains at their interface in solution. Interaction between the two domains of full-length hPin1 is absent when the protein is dissected into the catalytic and the WW domain. It indicates that the flexible linker, connecting both domains, promotes binding. By evaluation of NOESY spectra we can show that the ␣1/␤1 loop, which was proposed to undergo a large conformational rearrangement in the absence of sulfate and an Ala-Pro peptide, remained in the closed conformation under these conditions. Dissociation constants of 0.4 and 2.0 mM for sulfate and phosphate ions were measured at 12°C by fluorescence spectroscopy. Binding of sulfate prevents hPin1 aggregation and changes surface charges across the active center and around the reactive and catalytically essential Cys 113 . In the absence of sulfate and/or reducing agent this residue seems to promote aggregation, as observed in hPin1 solutions in vitro.

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Section: Methodsmentioning

confidence: 99%

Structural Analysis of the Mitotic Regulator hPin1 in Solution

Bayer

Goettsch

Mueller

et al. 2003

Journal of Biological Chemistry

118

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“…[77] A novel strategy for fast resonance assignment of 1 H, 15 N HSQC spectra based on pseudocontact shifts was also proposed. [78] Most of the data used in structural refinements involve [23,62,79] Since d pcs values are calculated as the difference between the chemical-shift values observed for the nuclei in a paramagnetic system and in a diamagnetic analogue, the latter is obtained by removing the paramagnetic ion, by substituting the paramagnetic metal ion with a diamagnetic metal, or by reducing the paramagnetic metal to a diamagnetic state.…”

Section: Hyperfine Shiftmentioning

confidence: 99%

“…In fact, studies that exploit a paramagnetic metal ion intentionally bound to a protein are many in the literature and are quite fashionable. [22][23][24][25][26][27][28][29][30][31][32][33][34][35] 2. Paramagnetism-Based Structural Restraints in Solution…”

Section: Introductionmentioning

confidence: 99%

NMR Spectroscopy of Paramagnetic Metalloproteins

et al. 2005

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This article deals with the solution structure determination of paramagnetic metalloproteins by NMR spectroscopy. These proteins were believed not to be suitable for NMR investigations for structure determination until a decade ago, but eventually novel experiments and software protocols were developed, with the aim of making the approach suitable for the goal and as user-friendly and safe as possible. In the article, we also give hints for the optimization of experiments with respect to each particular metal ion, with the aim of also providing a handy tool for nonspecialists. Finally, a section is dedicated to the significant progress made on 13C direct detection, which reduces the negative effects of paramagnetism and may constitute a new chapter in the whole field of NMR spectroscopy.

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“…In this case, a number of structural restraints based on paramagnetism are also available for solution-structure determination. [12,[33][34][35][36][37] Received: August 26, 2004 Revised: December 16, 2005 Published online: April 14, 2005 [*] Several methods have been proposed to remove J splitting from the acquisition dimension, such as band-selective homodecoupling, [10] maximum entropy reconstruction, [25] and other postacquisition methods. [26] We propose the method based on spinstate-selective schemes with the IPAP sequence [20,21] as an example, because it provides the best signal-to-noise ratio (S/N) in comparison with other methods (not shown).…”

mentioning

confidence: 99%

Complete Assignment of Heteronuclear Protein Resonances by Protonless NMR Spectroscopy

Bermel¹,

Bertini²,

Duma³

et al. 2005

Angew Chem Int Ed

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The complete assignment of the resonances of a protein is key to the determination of its solution structure by NMR spectroscopy and for the study of protein-protein and protein-ligand interactions. The proton-based assignment strategy usually starts with the correlation of individual resonances of each amino acid residue through scalar connectivities followed by linking them one after the other. [1,2] Although many different triple-resonance NMR spectroscopy experiments have been designed for full assignments, [2] spectral overlap can still lead to ambiguities. This poses a significant limiting factor in the cases of large and/or paramagnetic biomolecules. [3] After the pioneering report of 13 C NMR spin-system assignments of 13 C-enriched Anabaena 7120 ferredoxin by Markley and co-workers, [4] heteronuclear NMR spectroscopy experiments were progressively abandoned in favor of 1 Hdetection experiments. However, as was recently pointed out, heteronuclear NMR spectroscopy decreases the effect of detrimental transverse relaxation, which is typical of large or paramagnetic proteins. [5][6][7][8][9][10][11][12][13][14][15][16][17] For this reason, several heteronuclear NMR spectroscopy experiments for backbone assignment have been proposed for fully 13 C-and 15 N-enriched proteins. [13,14,17] Furthermore, backbone sequence-specific assignment by the recently-designed CANCO experiment has also been reported. [18] We present herein an extension of the set of exclusively heteronuclear experiments to protein side chain resonances for the complete heteronuclear assignment of a protein. With a novel CBCACO experiment the carbonyl carbon (CO) is linked to the C b and to the C a nuclei; the connection to the rest of the amino acid side chain is achieved through a 13 C-13 C TOCSY experiment with C a detection. In these experiments, we have successfully implemented spin-state selection methods for the removal of signal splitting in the acquisition dimension which is caused by multiple 13 C-13 C scalar couplings. This makes 13 C detection an amenable tool for high-resolution NMR spectroscopy. The proposed assignment strategy is summarized in Figure 1. A Figure 1. Illustration of the assignment procedure for 13 C NMR spectroscopy experiments. The assignment starts with analysis of the CACO experiment, which provides the correlation between the carbonyl carbon (CO) and the C a nuclei of each amino acid. The spin-system assignment is extended to the C b nuclei with the CBCACO experiment, and the process is completed with the TOCSY experiment, which provides correlation between the C a and the other carbon nuclei of the amino acid side chain. The amino acid spin systems are finally assigned in a sequence-specific manner with the aid of a CANCO experiment, [18] which provides the correlation of each CO to the two neighboring C a nuclei.

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Cited by 110 publications

References 28 publications

Structural Analysis of the Mitotic Regulator hPin1 in Solution

Structural Analysis of the Mitotic Regulator hPin1 in Solution

NMR Spectroscopy of Paramagnetic Metalloproteins

Complete Assignment of Heteronuclear Protein Resonances by Protonless NMR Spectroscopy

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