Pulse Sequences for Measurement of One-Bond 15N–1H Coupling Constants in the Protein Backbone

Lerche, Mathilde Hauge; Meißner, Axel; Poulsen, Flemming M.; Sørensen, Ole W.

doi:10.1006/jmre.1999.1820

Cited by 63 publications

(43 citation statements)

References 19 publications

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“…For sequential assignment the following spectra were recorded on the CT experiment was applied. 51 The spectra were processed using nmrPipe. 52 For comparison to the data by Keeler et al 20 a 13 C-NOESY-HSQC of the aliphatic region was recorded at pH 6.8, 100 mM NaCl and 298 K.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

Solution Structure of Human Prolactin

Teilum

Hoch

Goffin

et al. 2005

Journal of Molecular Biology

114

110

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We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/ placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactinreceptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.

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Section: Nmr Spectroscopymentioning

confidence: 99%

Solution Structure of Human Prolactin

Teilum

Hoch

Goffin

et al. 2005

Journal of Molecular Biology

114

110

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“…A suite of 2D [ 1 H, 15 N] correlation experiments have been proposed for different size proteins by choosing different components of multiplets caused by a 1 H-15 N coupling [55]. Fig.…”

Section: Measurement Of Residual Dipolar Couplings In Proteinsmentioning

confidence: 99%

“…The application of RDCs in combination with a comparative mutation study thus demonstrated the presence of precursors to the folding transition state under acid-unfolding conditions. The NH RDCs were measured from spin-state-selective (S 3 CT) filtered 1 H-15 N-HSQC experiments [41,55] (Sections 2.2 and 3.1), while C α -C , C -15 N, and C α -1 H α dipolar couplings were measured from 3D HNCO-based experiments [97,84] (Sections 3.2 and 3.7). The RDCs were extracted by frequency-displacement method.…”

Section: Native-like Topology Persistent In the Denatured Statementioning

confidence: 99%

Residual Dipolar Couplings: Measurements and Applications to Biomolecular Studies

Wang

2006

Annual Reports on NMR Spectroscopy

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Since the first successful demonstration of the tunable alignment of ubiquitin in an anisotropic liquid crystal medium only eight years ago, much progress has been made in both NMR pulse techniques for the measurements of various residual dipolar couplings (RDCs) in both proteins and nucleic acids, and applications of RDCs to many important problems in biochemistry and structural biology. In this annual report, we first review recent developments in NMR techniques for the measurements of many types of RDCs in both proteins and nucleic acids, especially a series of novel techniques for improving spectral resolution, signal to noise ratio and saving experimental time. We then describe the applications of RDCs to proteins including automated resonance assignment, structure determination, ligand-protein and proteinprotein dockings as well as protein folding.

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“…1 D NH RDCs were obtained from the peak displacement in the f2 dimension of two separate two-dimensional 1 H-15 N TROSY spectra (12). Values of 1 J NH obtained from isotropic phase control experiments lacking the Pf1 were subtracted from the peak splittings observed in the aligned phase spectra.…”

Section: Methodsmentioning

confidence: 99%

NMR Structure of the α-Hemoglobin Stabilizing Protein

Santiveri

Pérez‐Cañadillas

Vadivelu

et al. 2004

Journal of Biological Chemistry

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The structure of ␣-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free ␣-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three ␣-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/ trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to ␣-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer ␣ 1 ␤ 1 interface in tetrameric HbA.␣-Hemoglobin stabilizing protein (AHSP), 1 previously named EDRF (erythroid differentiation-related factor), was originally identified as a protein that showed a decrease in expression in the hematopoietic tissues of rodents and cattle infected with transmissible spongiform encephalopathies (1). EDRF was renamed AHSP after it was found that it binds specifically to ␣-hemoglobin in vitro (2). AHSP knock-out mice show erythrocyte abnormalities similar to those observed in ␤-thalassemia (2), and loss of AHSP aggravates the ␤-thalassemia phenotype in mice (38). ␤-Thalassemia is characterized by a reduction in the synthesis of ␤-globin chains. Consequently, the ␣-chains are in excess, and some of them precipitate, leading to defective red blood cells that determine the pathophysiology of ␤-thalassemia syndromes. The in vivo and in vitro data therefore both suggest that AHSP acts as a molecular chaperone for free ␣-hemoglobin (2, 3).Both AHSP and ␣-hemoglobin are monomeric in solution and bind each other with an association constant of 1 ϫ 10 7 M Ϫ1forming a complex with a 1:1 stoichiometry (4). The aim of the present study was to structurally characterize the AHSP-␣-hemoglobin interaction to understand the molecular basis of the AHSP function and its role as a hemoglobin chaperone. We have demonstrated by NMR that the AHSP native state is conformationally heterogeneous in solution and have identified the dynamic source of this behavior as a cis/trans isomerization of residue Pro-30. The slow time scale of this phenomenon allowed us to determine the high resolution three-dimensional structure of the two conformations of the wild-type AHSP under conditions where both conformations are equally populated. We were able to quench the c...

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Pulse Sequences for Measurement of One-Bond 15N–1H Coupling Constants in the Protein Backbone

Cited by 63 publications

References 19 publications

Solution Structure of Human Prolactin

Solution Structure of Human Prolactin

Residual Dipolar Couplings: Measurements and Applications to Biomolecular Studies

NMR Structure of the α-Hemoglobin Stabilizing Protein

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