Nuclear Magnetic Resonance 15N and 1H Resonance Assignments and Global Fold of Rusticyanin

Hunt, A. H.; Toy-Palmer, Anna; Assa‐Munt, Nuria; Cavanagh, John; Blake, Robert C.; Dyson, H. Jane

doi:10.1006/jmbi.1994.1737

Cited by 28 publications

(54 citation statements)

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“…For a fl-sheet protein such as rusticyanin, there is a risk in using only NOEs to establish sequential connectivities, since the d~y(i,i + 1) NOE is almost the only connection between spin systems; overlap in the C~H region can produce unacceptable ambiguities. Since ]~C-labeled rusticyanin was not available at that time, the ~H and ]SN assignments were confirmed using an 15N relayed-NOE experiment [5]. The present work demonstrates the validity of this approach: all of the sequential assignments made on the basis of the NOE and relayed-NOE data have now been confirmed by triple-resonance spectra.…”

Section: Resonance Assignmentssupporting

confidence: 56%

“…Triple-resonance and HCCH-TOCSY experiments were the most useful in determining aliphatic ]30 resonance assignments for rusticyanin, and the process was assisted by the availability of nearly-complete proton and ]~N resonance assignments [5]. The triple-resonance experiments also served to confirm the sequential assignments, which had relied heavily on inter-residue NOEs.…”

Section: Resonance Assignmentsmentioning

confidence: 99%

“…Although all of the backbone resonances with attached protons had been assigned previously using LSN spectra [5], a number of tH resonances remained unassigned, including those of the proline residues, which are not usually accessible in ~SN spectra because they have no amide proton. Complete 130 assignments were obtained from the CBCA(CO)NH and C(CO)NH-TOCSY spectra for all of the proline residues in rusticyanin except for Pro 94, which is followed by another proline, and IH assignments for the C~H and CPH were obtained unambiguously from the HBHA(CO)NH experiment.…”

Section: H Assignments Fi~r Proline Residuesmentioning

confidence: 99%

“…For almost all Type 1 copper proteins the copper is labile under these conditions, due to protonation of one or both of the histidine ligands [4]. Unusual copper ligands can apparently be ruled out, since rusticyanin appears to contain the classic Type 1 copper site (His2 Cys Met) [5,6]. It therefore appears that the structure of the protein *Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

“…Assignment of the ~H and 15N NMR spectra of reduced (Cu(I)) rusticyanin has been largely completed using ~SN-labeled protein obtained from Thiobacillus [5]. Excellent yields of protein uniformly labeled with ~3C and ~SN have been obtained from the over-expression in Escherichia coli of a synthetic gene [6].…”

Section: Introductionmentioning

confidence: 99%

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Complete ¹³C assignments for recombinant Cu(I) rusticyanin prediction of secondary structure from patterns of chemical shifts

1995

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Complete resonance assignments for the ~sC spectrum of reduced (Cn(I)) rusticyanin have been made using 13C,~SN doubly labeled recombinant material. The reported assignments include those for the earboxyl and carbonyl carbon atoms and protonated aromatic ring carbons, and were obtained using a variety of 2-and 3D inverse-detected NMR experiments, including 13C,ISN,IH triple resonance experiments and HCCH-COSY and -TOCSY. Backbone carbonyl assignments were obtained using 3D HNCO and HCACO spectra, and modified versions of 2D H(CA)CO and HMBC spectra were used to obtain side-chain carboxyl carbon and methionine z-methyl carbon assignments, respectively. A comparison of the 13C~, ~5C° and 13CO chemical shifts with published 'random coil' values confirms the conclusion reached from a consideration of the 3JnN ~ coupling constants and the pattern of sequential NOEs, that the protein consists largely of B-structure.

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Section: Resonance Assignmentssupporting

confidence: 56%

Section: Resonance Assignmentsmentioning

confidence: 99%

Section: H Assignments Fi~r Proline Residuesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Complete ¹³C assignments for recombinant Cu(I) rusticyanin prediction of secondary structure from patterns of chemical shifts

1995

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Electrostatic Effects on the Thermodynamics of Protonation of Reduced Plastocyanin

et al. 2005

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The L12E, L12K, Q88E, and Q88K variants of spinach plastocyanin have been electrochemically investigated. The effects of insertion of net charges near the metal site on the thermodynamics of protonation and detachment from the copper(I) ion of the His87 ligand have been evaluated. The mutation-induced changes in transition enthalpy cannot be explained by electrostatic considerations. The existence of enthalpy/entropy (H/S) compensation within the protein series indicates that solvent-reorganization effects control the differences in transition thermodynamics. Once these compensating contributions are factorized out, the resulting modest differences in transition enthalpies turn out to be those that can be expected on purely electrostatic grounds. Therefore, this work shows that the acid transition in cupredoxins involves a reorganization of the H-bonding network within the hydration sphere of the molecule in the proximity of the metal center that dominates the observed transition thermodynamics and masks the differences that are due to protein-based effects.

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Heterogeneity of the covalent structure of the blue copper protein umecyanin from horseradish roots

et al. 1995

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The covalent structure of umecyanin has been determined by a combination of classical Edman degradation sequence analysis and plasma desorption, laser desorption, and electrospray ionization mass spectrometry. The preparation appeared to contain two isoforms having either a valine (75%) or an isoleucine (25%) residue at position 48. The polypeptide chain of 115 amino acids is strongly heterogeneous at its C-terminal end as a result of proteolytic cleavages at several places within the last 10 residues. The major fraction of the umecyanin preparation is only 106 residues long. The C-terminal tail 107-115 contains mainly alanine and glycine residues and a single hydroxyproline residue. In the native protein there is a disulfide bridge between Cys 91 and Cys 57, but in the apoprotein there is a disulfide shift that involves Cys 91 and one of the four copper binding residues (Cys 85). The three other ligand binding residues are His 44, His 90, and Gln 95. This tetrad of amino acids is the same as occurs in other type 1 copper proteins from plants such as cucumber peeling cupredoxin and lacquer tree stellacyanin. The umecyanin isoforms are glycoproteins with a glycan core having the same carbohydrate composition as that of horseradish peroxidase, a fact that is convincingly supported thanks to the high accuracy of the electrospray mass spectrometric technique. We suggest that the glycan may play a role in the association of the protein to the cellular membrane, but the precise functional role of umecyanin remains to be determined. We also discuss the evolutionary position of umecyanin in relation to the type 1 copper proteins in general.

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Nuclear Magnetic Resonance 15N and 1H Resonance Assignments and Global Fold of Rusticyanin

Cited by 28 publications

References 0 publications

Complete ¹³C assignments for recombinant Cu(I) rusticyanin prediction of secondary structure from patterns of chemical shifts

Complete ¹³C assignments for recombinant Cu(I) rusticyanin prediction of secondary structure from patterns of chemical shifts

Electrostatic Effects on the Thermodynamics of Protonation of Reduced Plastocyanin

Heterogeneity of the covalent structure of the blue copper protein umecyanin from horseradish roots

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Nuclear Magnetic Resonance 15N and 1H Resonance Assignments and Global Fold of Rusticyanin

Cited by 28 publications

References 0 publications

Complete 13C assignments for recombinant Cu(I) rusticyanin prediction of secondary structure from patterns of chemical shifts

Complete 13C assignments for recombinant Cu(I) rusticyanin prediction of secondary structure from patterns of chemical shifts

Electrostatic Effects on the Thermodynamics of Protonation of Reduced Plastocyanin

Heterogeneity of the covalent structure of the blue copper protein umecyanin from horseradish roots

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Complete ¹³C assignments for recombinant Cu(I) rusticyanin prediction of secondary structure from patterns of chemical shifts

Complete ¹³C assignments for recombinant Cu(I) rusticyanin prediction of secondary structure from patterns of chemical shifts