Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins

Kay, Lewis E.; Ikura, Mitsuhiko; Tschudin, Rolf; Bax, Ad

doi:10.1016/0022-2364(90)90333-5

Cited by 451 publications

(510 citation statements)

References 21 publications

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“…Abundant spin exchange occurring among nearby 1 H nuclei in model peptides (22), and dilute spin exchange among 15 N sites in both model peptides and proteins (20,21) have been demonstrated. Alternative assignment strategies that utilize uniformly 13 C and 15 Nlabeled proteins (41), analogous to those used in solution NMR spectroscopy (42), are also under development.…”

Section: Resultsmentioning

confidence: 99%

Complete resolution of the solid-state NMR spectrum of a uniformly ¹⁵ N-labeled membrane protein in phospholipid bilayers

Marassi

Ramamoorthy

Opella

1997

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

204

180

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Section: Resultsmentioning

confidence: 99%

Complete resolution of the solid-state NMR spectrum of a uniformly ¹⁵ N-labeled membrane protein in phospholipid bilayers

Marassi

Ramamoorthy

Opella

1997

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

204

180

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“…All NMR spectra were processed using NMRPipe (40) and analyzed using NMRView (41). Two-dimensional 15 N-and 13 C-edited heteronuclear single quantum coherence (HSQC) and threedimensional HNCA, HNCACB, HNCO, HN(CA)CO, HBHA-(CO)NH, CBCA(CO)NH, (H)CC(CO)NH-TOCSY, (H)CCH-COSY, and (H)CCH-TOCSY experiments were performed to obtain the chemical shift assignments of backbone and side chain atoms (42)(43)(44)(45)(46)(47). The three-dimensional 15 N-and 13 C-edited NOESY-HSQC spectra (mixing time 100 ms) were collected to confirm the chemical shift assignments and generate distance restraints for structure calculations (48).…”

Section: Methodsmentioning

confidence: 99%

Solution Structures and Backbone Dynamics of a Flavodoxin MioC from Escherichia coli in both Apo- and Holo-forms

Zhang

et al. 2006

Journal of Biological Chemistry

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Flavodoxins play central roles in the electron transfer involving various biological processes in microorganisms. The mioC gene of Escherichia coli encodes a 16-kDa flavodoxin and locates next to the chromosomal replication initiation origin (oriC). Extensive researches have been carried out to investigate the relationship between mioC transcription and replication initiation. Recently, the MioC protein was proposed to be essential for the biotin synthase activity in vitro. Nevertheless, the exact role of MioC in biotin synthesis and its physiological function in vivo remain elusive. In order to understand the molecular basis of the biological functions of MioC and the cofactor-binding mechanisms of flavodoxins, we have determined the solution structures of both the apo-and holo-forms of E. coli MioC protein at high resolution by nuclear magnetic resonance spectroscopy. The overall structures of both forms consist of an ␣/␤ sandwich, which highly resembles the classical flavodoxin fold. However, significant diversities are observed between the two forms, especially the stabilization of the FMN-binding loops and the notable extension of secondary structures upon FMN binding. Structural comparison reveals fewer negative charged and aromatic residues near the FMN-binding site of MioC, as compared with that of flavodoxin 1 from E. coli, which may affect both the redox potentials and the redox partner interactions. Furthermore, the backbone dynamics studies reveal the conformational flexibility at different time scales for both apo-and holo-forms of MioC, which may play important roles for cofactor binding and electron transfer.Flavodoxins are small FMN-binding proteins found mainly in prokaryotes that are involved in the electron transfer chain reactions of various biological processes, including photosynthesis (1, 2), nitrate reduction (3), methionine synthesis (4 -7), biotin synthesis (8 -11), and activation of certain enzymes, such as pyruvate-formate lyase (13) and (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate synthase (14). It has been established by potentiometry that electrons flow from NADPH to flavodoxin reductase and then to flavodoxin, which subsequently transfers the electron to the downstream targets (15). Eukaryotes and most prokaryotes also contain proteins carrying domains homologous to flavodoxins and flavodoxin reductase in a single polypeptide chain, such as the NADPH-cytochrome P450 oxidoreductase (CPR) 2 (16, 17), nitric-oxide synthase (NOS) (18), methionine synthase reductase (19), and sulfite reductase (20,21). These domains function in a similar fashion as the flavodoxins and flavodoxin reductase proteins in prokaryotic cells. Extensive efforts have been devoted to understanding the cofactor binding, redox partner interaction, and electron transfer mechanisms of flavodoxins and flavodoxinlike domains as well as the folding and stability of these proteins (22-26). However, the detailed mechanisms are not clear yet and remain to be further elucidated.Flavodoxins are classified into the long-...

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“…All NMR experiments ( H HSQC, two-dimensional HNCO, three-dimensional HNCA, two-dimensional HCCH, and two-dimensional (H␤)C␤(C␥C␦)H␦) were carried out by a Bruker ARX-500 and/or AMX-500 spectrometer at 25°C. The NMR parameters for this histidine study and the references for basic pulse sequences (31)(32)(33)(34)(35)(36)(37) (38).…”

Section: Isotope Labeling Of Hcaii-to Detect Imidazolementioning

confidence: 99%

Tautomerism of Histidine 64 Associated with Proton Transfer in Catalysis of Carbonic Anhydrase

Shimahara

Yoshida

Shibata

et al. 2007

Journal of Biological Chemistry

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. These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His 64 can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time-or energy-consuming processes.Carbonic anhydrase (CA) 2 (EC 4.2.1.1) is a ubiquitous enzyme that catalyzes the reversible hydration of carbon dioxide (1). Isozymes of carbonic anhydrase regulate or function in such diverse physiological processes as pH regulation, ion transport, water-electrolyte balance, bicarbonate secretion-absorption, bone resorption, maintenance of intraocular pressure, renal acidification, and brain development (2). Nonfunctioning CA is implicated in such diseases as osteopetrosis syndrome, glaucoma, respiratory acidosis, epilepsy, and Méni-ère syndrome. Diseases due to CA deficiency include those affecting bones, the brain, and the kidneys. Consequently determining the detailed structure/function relationships or mechanisms responsible for its catalytic properties is mandatory for developing inhibitors or replacement therapies.CA is present in at least three gene families (␣, ␤, and ␥), which has made it a popular model for the study of the evolution of gene families and protein folding, and for transgenic and gene target studies (2). Among the three families, the ␣ family is the best characterized, with 11 known isozymes identified in mammals. Earnhardt and co-workers have summarized maximal k cat and k cat /K m values for CO 2 hydration by isozyme I-VII (3). The human isozyme II (hCAII) has a remarkably high turnover rate or catalytic efficiency (k cat /K m ϭ 1.5 ϫ 10 8 M Ϫ1 s Ϫ1 ) that is very close to the frequency with which the enzyme and substrate molecules collide with each other in solution.It is widely accepted that the hydration of CO 2 catalyzed by hCAII proceeds through several chemical steps as shown in Scheme 1 (1, 4, 5): the direct nucleophilic attack of the zinc-bound hydroxide ion on the carbonyl carbon of substrate CO 2 (structures 1-2), the formation of a zinc-bound bicarbonate intermediate (structures 2-3), the isomerization of the bicarbonate ion (structures 3-4), the exchange of the product bicarbonate ion with a H 2 O (structures 4 -5), and the regeneration of the zinc-bound hydroxide ion by the transfer of a proton to bulk solvent (structures 1-5). The proton transfer step (structures 1-5) consists of two substeps: 1) an intra-molecular transfer of protons to another residue in the enzyme and 2) a release of protons to the outside of the enzyme with the aid of a base. The intra-molecular proton transfer is the rate-limiting step of the maximal turnover rate (10 6 s Ϫ1 ) at high concentrations of a base, whereas the proton release into the medium is rate-limiting at low buffer concentrations.

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Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins

Cited by 451 publications

References 21 publications

Complete resolution of the solid-state NMR spectrum of a uniformly ¹⁵ N-labeled membrane protein in phospholipid bilayers

Complete resolution of the solid-state NMR spectrum of a uniformly ¹⁵ N-labeled membrane protein in phospholipid bilayers

Solution Structures and Backbone Dynamics of a Flavodoxin MioC from Escherichia coli in both Apo- and Holo-forms

Tautomerism of Histidine 64 Associated with Proton Transfer in Catalysis of Carbonic Anhydrase

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Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins

Cited by 451 publications

References 21 publications

Complete resolution of the solid-state NMR spectrum of a uniformly 15 N-labeled membrane protein in phospholipid bilayers

Complete resolution of the solid-state NMR spectrum of a uniformly 15 N-labeled membrane protein in phospholipid bilayers

Solution Structures and Backbone Dynamics of a Flavodoxin MioC from Escherichia coli in both Apo- and Holo-forms

Tautomerism of Histidine 64 Associated with Proton Transfer in Catalysis of Carbonic Anhydrase

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Complete resolution of the solid-state NMR spectrum of a uniformly ¹⁵ N-labeled membrane protein in phospholipid bilayers

Complete resolution of the solid-state NMR spectrum of a uniformly ¹⁵ N-labeled membrane protein in phospholipid bilayers