Biomedical subjects

Marcellus Ubbink

Publications and source records attributed to Marcellus Ubbink.

At least 19 recordsLinked to original sources

PubMed2007

Redox-state-dependent complex formation between pseudoazurin and nitrite reductase.

Bacterial copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide as part of the denitrification process. Pseudoazurin interacts with nitrite reductase in a transient fashion to supply the necessary electrons. The redox-state dependence of complex formation between pseudoazurin and nitrite reductase was studied by nuclear magnetic resonance spectroscopy and isothermal titration calorimetry. Binding of pseudoazurin in the reduced state is characterized by the presence of two binding modes, a slow and a fast exchange mode, with a K(d)(app) of 100 microM. In the oxidized state of pseudoazurin, binding occurs in a single fast exchange mode with a similar affinity. Metal-substituted proteins have been used to show that the mode of binding of pseudoazurin is independent of the metal charge of nitrite reductase. Contrary to what was found for other cupredoxins, protonation of the exposed His ligand to the copper of pseudoazurin, His81, does not appear to be involved directly in the dual binding mode of the reduced form. A model assuming the presence of a minor form of pseudoazurin is proposed to explain the behavior of the complex in the reduced state.

Alcaligenes faecalis↗

PubMed2007

Long-range-distance NMR effects in a protein labeled with a lanthanide-DOTA chelate.

A two-thiol reactive lanthanide-DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) chelate, CLaNP-3 (CLaNP=caged lanthanide NMR probe), was synthesized for the rigid attachment to cysteine groups on a protein surface, and used to obtain long-range-distance information from the {15N,1H} HSQC spectra of the protein-lanthanide complex. The DOTA ring exhibits several isomers that are in exchange; however, single resonances were observed for most amide groups in the protein, allowing determination of a single, apparent magnetic-susceptibility tensor. Pseudocontact shifts caused by Yb-containing CLaNP-3 were observed for atoms at 15-35 A from the metal. By using Gd-containing CLaNP-3, relaxation effects were observed, allowing distances up to 30 A from the paramagnetic center to be determined accurately. Similar results were obtained with a Gd-DTPA (diethylene-triaminepentaacetic acid) chelate, CLaNP-1, bound in the same bidentate manner to the protein. This study demonstrates that bidentate attachment of a paramagnetic probe enables determination of long-range distances.

Chelating Agents↗

PubMed2006

Solution structure and dynamics of the complex between cytochrome c and cytochrome c peroxidase determined by paramagnetic NMR.

The physiological complex of yeast cytochrome c peroxidase and iso-1-cytochrome c is a paradigm for biological electron transfer. Using paramagnetic NMR spectroscopy, we have determined the conformation of the protein complex in solution, which is shown to be very similar to that observed in the crystal structure [Pelletier H, Kraut J (1992) Science 258:1748-1755]. Our results support the view that this transient electron transfer complex is dynamic. The solution structure represents the dominant protein-protein orientation, which, according to our estimates, is occupied for >70% of the lifetime of the complex, with the rest of the time spent in the dynamic encounter state. Based on the observed paramagnetic effects, we have delineated the conformational space sampled by the protein molecules during the dynamic part of the interaction, providing experimental support for the theoretical predictions of the classical Brownian dynamics study [Northrup SH, Boles JO, Reynolds JCL (1988) Science 241:67-70]. Our findings corroborate the dynamic behavior of this complex and offer an insight into the mechanism of the protein complex formation in solution.

Cytochrome-c Peroxidase↗

PubMed2006

Ferredoxin/ferredoxin-thioredoxin reductase complex: Complete NMR mapping of the interaction site on ferredoxin by gallium substitution.

The reduction of ferredoxin-thioredoxin reductase (FTR) by plant-type ferredoxin plays an important role in redox regulation in plants and cyanobacteria. Nuclear magnetic resonance (NMR) was used to map the binding sites on Synechocystis ferredoxin for FTR. A gallium-substituted structural analog of this [2Fe-2S] ferredoxin was obtained by reconstituting the apoprotein in a refolding buffer containing gallium. For the first time, the complete interaction interface of a [2Fe-2S] ferredoxin with a target enzyme has been mapped by NMR chemical shift perturbation with this diamagnetic structural analog.

Amino Acid Sequence↗

PubMed2006

Spectroscopic characterization of a high-potential lipo-cupredoxin found in Streptomyces coelicolor.

For many streptomycetes, a distinct dependence on the "bioavailability" of copper ions for their morphological development has been reported. Analysis of the Streptomyces coelicolor genome reveals a number of gene products encoding for putative copper-binding proteins. One of these appears as an unusual copper-binding protein with a lipoprotein signal sequence and a cupredoxin-like domain harboring a putative Type-1 copper-binding motif. Cloning of this gene from S. coelicolor and subsequent heterologous expression in Escherichia coli has allowed for a thorough spectroscopic interrogation of this putative copper-binding protein. Optical and electron paramagnetic resonance spectroscopies have confirmed the presence of a "classic" Type-1 copper site with the axial ligand to the copper a methionine. Paramagnetic NMR spectroscopy on both the native Cu(II) form and Co(II)-substituted protein has yielded active-site structural information, which on comparison with that of other cupredoxin active sites reveals metal-ligand interactions most similar to the "classic" Type-1 copper site found in the amicyanin family of cupredoxins. Despite this high structural similarity, the Cu(II)/(I) midpoint potential of the S. coelicolor protein is an unprecedented +605 mV vs normal hydrogen electrode at neutral pH (amicyanin approximately +250 mV), with no active-site protonation of the N-terminal His ligand observed. Suggestions for the physiological role/function of this high-potential cupredoxin are discussed.

Amino Acid Sequence↗

PubMed2006

Conformational changes during apoplastocyanin folding observed by photocleavable modification and transient grating.

A new method to investigate the initial protein folding dynamics is developed based on a pulsed laser light triggering method and a unique transient grating method. The side chain of the cysteine residue of apoplastocyanin (apoPC) was site-specifically modified with a 4,5-dimethoxy-2-nitrobenzyl derivative, where the CD and 2D NMR spectra showed that the modified apoPC was unfolded. The substituent was cleaved with a rate of about 400 ns by photoirradiation, which was monitored by the disappearance of the absorption band at 355 nm and the increase in the transient grating signal. After a sufficient time from the photocleavage reaction, the CD and NMR spectra showed that the native beta-sheet structure was recovered. Protein folding dynamics was monitored in the time domain with the transient grating method from a viewpoint of the molecular volume change and the diffusion coefficient, both of which reflect the global structural change, including the protein-water interaction. The observed volume decrease of apoPC with a time scale of 270 micros is ascribed to the initial hydrophobic collapse. The increase in the diffusion coefficient (23 ms) is considered to indicate a change from an intermolecular to an intramolecular hydrogen bonding network. The initial folding process of apoPC is discussed based on these observations.

Apoproteins↗

PubMed2006

High-field (275 GHz) spin-label EPR for high-resolution polarity determination in proteins.

The polarity of protein surfaces is one of the factors driving protein-protein interactions. High-field, spin-label EPR at 95 GHz, i.e., 10 times higher than conventional EPR, is an upcoming technique to determine polarity parameters of the inside of proteins. Here we show that by 275 GHz EPR even the small polarity differences of sites at the protein surface can be discriminated. To do so, four single cysteine mutations were introduced at surface sites (positions 12, 27, 42, and 118) of azurin and spin labeled. By 275 GHz EPR in frozen solution, polarity/proticity differences between all four sites can be resolved, which is impossible by 95 GHz EPR. In addition, by 275 GHz EPR, two spectral components are observed for all mutants. The difference between them corresponds to one additional hydrogen bond.

Azurin↗

PubMed2005

An NMR-based docking model for the physiological transient complex between cytochrome f and cytochrome c6.

The physiological transient complex between cytochrome f (Cf) and cytochrome c(6) (Cc(6)) from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by NMR spectroscopy. The binding constant at low ionic strength is 8 +/- 2 mM(-1), and the binding site of Cc(6) for Cf is localized around its exposed haem edge. On the basis of the experimental data, the resulting docking simulations suggest that Cc(6) binds to Cf in a fashion that is analogous to that of plastocyanin but differs between prokaryotes and eukaryotes.

Cytochromes c6↗

PubMed2005

Different modes of interaction in cyanobacterial complexes of plastocyanin and cytochrome f.

The highly efficient electron-transfer chain in photosynthesis demonstrates a remarkable variation among organisms in the type of interactions between the soluble electron-transfer protein plastocyanin and it partner cytochrome f. The complex from the cyanobacterium Nostoc sp. PCC 7119 was studied using nuclear magnetic resonance spectroscopy and compared to that of the cyanobacterium Phormidium laminosum. In both systems, the main site of interaction on plastocyanin is the hydrophobic patch. However, the interaction in the Nostoc complex is highly dependent on electrostatics, contrary to that of Phormidium, resulting in a binding constant that is an order of magnitude larger at low ionic strength for the Nostoc complex. Studies of the mixed complexes show that these differences in interactions are mainly attributable to the surface properties of the plastocyanins.

Cyanobacteria↗

PubMed2005

Structure of the complex between plastocyanin and cytochrome f from the cyanobacterium Nostoc sp. PCC 7119 as determined by paramagnetic NMR. The balance between electrostatic and hydrophobic interactions within the transient complex determines the relative orientation of the two proteins.

The complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc has been characterized by NMR spectroscopy. The binding constant is 16 mM(-1), and the lifetime of the complex is much less than 10 ms. Intermolecular pseudo-contact shifts observed for the plastocyanin amide nuclei, caused by the heme iron, as well as the chemical-shift perturbation data were used as the sole experimental restraints to determine the orientation of plastocyanin relative to cytochrome f with a precision of 1.3 angstroms. The data show that the hydrophobic patch surrounding tyrosine 1 in cytochrome f docks the hydrophobic patch of plastocyanin. Charge complementarities are found between the rims of the respective recognition sites of cytochrome f and plastocyanin. Significant differences in the relative orientation of both proteins are found between this complex and those previously reported for plants and Phormidium, indicating that electrostatic and hydrophobic interactions are balanced differently in these complexes.

Animals↗

PubMed2005

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK.

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.

Animals↗

PubMed2005

The effects of ligand exchange and mobility on the peroxidase activity of a bacterial cytochrome c upon unfolding.

The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant.

Amino Acid Substitution↗

PubMed2005

Pseudoazurin-nitrite reductase interactions.

The nitrite reductase-binding site on pseudoazurin has been determined by using NMR chemical-shift perturbations. It comprises residues in the hydrophobic patch surrounding the exposed copper ligand His81 as well as several positively charged residues. The binding site is similar for both redox states of pseudoazurin, despite differences in the binding mode. The results suggest that pseudoazurin binds in a well-defined orientation. Docking simulations provide a putative structure of the complex with a binding site on nitrite reductase that has several hydrophobic and polar residues as well as a ridge of negatively charged side chains and a copper-to-copper distance of 14 A.

Azurin↗

PubMed2005

Solution structure of the second PDZ domain of the neuronal adaptor X11alpha and its interaction with the C-terminal peptide of the human copper chaperone for superoxide dismutase.

Protection against reactive oxygen species is provided by the copper containing enzyme superoxide dismutase 1 (SOD1). The copper chaperone CCS is responsible for copper insertion into apo-SOD1. This role is impaired by an interaction between the second PDZ domain (PDZ2alpha) of the neuronal adaptor protein X11alpha and the third domain of CCS (McLoughlin et al. (2001) J. Biol. Chem., 276, 9303-9307). The solution structure of the PDZ2alpha domain has been determined and the interaction with peptides derived from CCS has been explored. PDZ2alpha binds to the last four amino acids of the CCS protein (PAHL) with a dissociation constant of 91 +/- 2 microM. Peptide variants have been used to map the interaction areas on PDZ2alpha for each amino acid, showing an important role for the C-terminal leucine, in line with canonical PDZ-peptide interactions.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The effect of replacing the axial methionine ligand with a lysine residue in cytochrome c-550 from Paracoccus versutus assessed by X-ray crystallography and unfolding.

The structure of cytochrome c-550 from the nonphotosynthetic bacteria Paraccocus versutus has been solved by X-ray crystallography to 1.90 A resolution, and reveals a high structural homology to other bacterial cytochromes c(2). The effect of replacing the axial heme-iron methionine ligand with a lysine residue on protein structure and unfolding has been assessed using the M100K variant. From X-ray structures at 1.95 and 1.55 A resolution it became clear that the amino group of the lysine side chain coordinates to the heme-iron. Structural differences compared to the wild-type protein are confined to the lysine ligand loop connecting helices four and five. In the heme cavity an additional water molecule is found which participates in an H-bonding interaction with the lysine ligand. Under cryo-conditions extra electron density in the lysine ligand loop is revealed, leading to residues K97 to T101 being modeled with a double main-chain conformation. Upon unfolding, dissociation of the lysine ligand from the heme-iron is shown to be pH dependent, with NMR data consistent with the occurrence of a ligand exchange mechanism similar to that seen for the wild-type protein.

Bacterial Proteins↗

PubMed2004

The transient complex of poplar plastocyanin with cytochrome f: effects of ionic strength and pH.

The orientation of poplar plastocyanin in the complex with turnip cytochrome f has been determined by rigid-body calculations using restraints from paramagnetic NMR measurements. The results show that poplar plastocyanin interacts with cytochrome f with the hydrophobic patch of plastocyanin close to the heme region on cytochrome f and via electrostatic interactions between the charged patches on both proteins. Plastocyanin is tilted relative to the orientation reported for spinach plastocyanin, resulting in a longer distance between iron and copper (13.9 A). With increasing ionic strength, from 0.01 to 0.11 M, all observed chemical-shift changes decrease uniformly, supporting the idea that electrostatic forces contribute to complex formation. There is no indication for a rearrangement of the transient complex in this ionic strength range, contrary to what had been proposed earlier on the basis of kinetic data. By decreasing the pH from pH 7.7 to pH 5.5, the complex is destabilized. This may be attributed to the protonation of the conserved acidic patches or the copper ligand His87 in poplar plastocyanin, which are shown to have similar pK(a) values. The results are interpreted in a two-step model for complex formation.

Brassica napus↗

PubMed2004

A caged lanthanide complex as a paramagnetic shift agent for protein NMR.

A lanthanide complex, named CLaNP (caged lanthanide NMR probe) has been developed for the characterisation of proteins by paramagnetic NMR spectroscopy. The probe consists of a lanthanide chelated by a derivative of DTPA (diethylenetriaminepentaacetic acid) with two thiol reactive functional groups. The CLaNP molecule is attached to a protein by two engineered, surface-exposed, Cys residues in a bidentate manner. This drastically limits the dynamics of the metal relative to the protein and enables measurements of pseudocontact shifts. NMR spectroscopy experiments on a diamagnetic control and the crystal structure of the probe-protein complex demonstrate that the protein structure is not affected by probe attachment. The probe is able to induce pseudocontact shifts to at least 40 A from the metal and causes residual dipolar couplings due to alignment at a high magnetic field. The molecule exists in several isomeric forms with different paramagnetic tensors; this provides a fast way to obtain long-range distance restraints.

Azurin↗

PubMed2004

Mapping of the binding site on pseudoazurin in the transient 152 kDa complex with nitrite reductase.

Nitrite reductase (NiR) catalyzes the reduction of nitrite to nitrite oxide as a part of the denitrification process. In Alcaligenes faecalis S-6, the copper protein pseudoazurin acts as electron donor to NiR. The binding surface of pseudoazurin involved in the formation of the 152 kDa complex with NiR has been determined by NMR using cross saturation from NiR to perdeuterated pseudoazurin. Due to the transient nature of the complex, saturation effects can be observed on the resonances of the unbound protein. The binding site comprises the hydrophobic area surrounding the exposed copper ligand His81, suggesting that this residue is important for efficient electron transfer.

Alcaligenes faecalis↗