Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge

The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10^- 5 M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-β fold formed of two β-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal β-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal β-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal β-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-β fold and the methionine-rich Cu(I)-binding site.     

High-resolution structure determination of the CylR2 homodimer using paramagnetic relaxation enhancement and structure-based prediction of molecular alignment

Structure determination of homooligomeric proteins by NMR spectroscopy is difficult due to the lack of chemical shift perturbation data, which is very effective in restricting the binding interface in heterooligomeric systems, and the difficulty of obtaining a sufficient number of intermonomer distance restraints. Here we solved the high-resolution solution structure of the 15.4 kDa homodimer CylR2, the regulator of cytolysin production from Enterococcus faecalis, which deviates by 1.1 angstroms from the previously determined X-ray structure. We studied the influence of different experimental information such as long-range distances derived from paramagnetic relaxation enhancement, residual dipolar couplings, symmetry restraints and intermonomer Nuclear Overhauser Effect restraints on the accuracy of the derived structure. In addition, we show that it is useful to combine experimental information with methods of ab initio docking when the available experimental data are not sufficient to obtain convergence to the correct homodimeric structure. In particular, intermonomer distances may not be required when residual dipolar couplings are compared to values predicted on the basis of the charge distribution and the shape of ab initio docking solutions.     

Vivaldi: Visualization and validation of biomacromolecular NMR structures from the PDB

We describe Vivaldi (VIsualization and VALidation DIsplay; http://pdbe.org/vivaldi ), a web‐based service for the analysis, visualization, and validation of NMR structures in the Protein Data Bank (PDB). Vivaldi provides access to model coordinates and several types of experimental NMR data using interactive visualization tools, augmented with structural annotations and model‐validation information. The service presents information about the modeled NMR ensemble, validation of experimental chemical shifts, residual dipolar couplings, distance and dihedral angle constraints, as well as validation scores based on empirical knowledge and databases. Vivaldi was designed for both expert NMR spectroscopists and casual non‐expert users who wish to obtain a better grasp of the information content and quality of NMR structures in the public archive. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Structural dynamics of a single-stranded RNA–helix junction using NMR

Many regulatory RNAs contain long single strands (ssRNA) that adjoin secondary structural elements. Here, we use NMR spectroscopy to study the dynamic properties of a 12-nucleotide (nt) ssRNA tail derived from the prequeuosine riboswitch linked to the 3′ end of a 48-nt hairpin. Analysis of chemical shifts, NOE connectivity, ¹³C spin relaxation, and residual dipolar coupling data suggests that the first two residues (A25 and U26) in the ssRNA tail stack onto the adjacent helix and assume an ordered conformation. The following U26-A27 step marks the beginning of an A₆-tract and forms an acute pivot point for substantial motions within the tail, which increase toward the terminal end. Despite substantial internal motions, the ssRNA tail adopts, on average, an A-form helical conformation that is coaxial with the helix. Our results reveal a surprising degree of structural and dynamic complexity at the ssRNA–helix junction, which involves a fine balance between order and disorder that may facilitate efficient pseudoknot formation on ligand recognition.     

Realistic Ensemble Models of Intrinsically Disordered Proteins Using a Structure-Encoding Coil Database

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A diagnostic for collaborative monitoring in forest landscape restoration

Monitoring is crucial to meet the goals of the major global forest landscape restoration (FLR) initiatives that are underway. If members of the global FLR community are going to learn from one another, a multi‐scalar, multi‐site monitoring approach is needed to generate information that can provide the basis for social learning and adaptive management, both of which are essential processes for FLR. This requires reframing and expanding the perspective of monitoring so that compliance monitoring is just one component of a multidimensional approach where collaborative monitoring and compliance‐oriented monitoring are complementary. However, FLR planners and implementers often lack experience in applying collaborative approaches in multi‐stakeholder settings, and there are few tools that show how to implement FLR or to engage in collaborative monitoring in FLR. Through a literature review, we identified the factors that contribute to successful collaborative monitoring in FLR and synthesized them into a diagnostic that was vetted by 20 global experts. The result is a checklist of 42 core success factors to be assessed at local, subnational, and national levels at different stages in the planning and implementation of FLR. The tool has practical application by providing guidance on best practices: specifically, how to start collaborative monitoring, and more generally, how to plan, prepare for, and evaluate FLR activities. This diagnostic complements other diagnostics, such as those used to identify FLR sites, as it can identify preexisting strengths and weaknesses in new initiatives, or pinpoint problems with ongoing implementation. The diagnostic explicitly addresses issues of scale, including multiple sites, governance levels, and changes over time.     

Directly observed hydrogen bonds at calcium-binding-sites of calmodulin in solution relate to affinity of the calcium-binding

Molecular tuning to calcium-binding in the EF-hand motif of holo-calmodulin was studied in solution by NMR ^h3J_NC′ H-bond couplings. In the N-terminus lobe of holo-calmodulin, the glutamate crucial for Ca²⁺ coordination has network of H-bonds weaker than inferred from the X-ray crystal structure. This glutamate at position 12 appears shifted away from the Ca²⁺ preferred coordination, which can explain the lower affinity of the calcium-binding to the N-terminus with respect to C-terminus EF hands.     

Intermolecular dynamics studied by paramagnetic tagging

Yeast cytochrome c and bovine adrenodoxin form a dynamic electron transfer complex, which is a pure encounter complex. It is demonstrated that the dynamic nature of the interaction can readily be probed by using a rigid lanthanide tag attached to cytochrome c. The tag, Caged Lanthanide NMR Probe 5, induces pseudocontact shifts and residual dipolar couplings and does not perturb the binding interface. Due to the dynamics in the complex, residual dipolar couplings in adrenodoxin are very small. Simulation shows that cytochrome c needs to sample a large part of the surface of adrenodoxin to explain the small degree of alignment observed for adrenodoxin. The applied method provides a simple and straightforward way to observe dynamics in protein complexes or domain–domain mobility without the need for external alignment media. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural characterization of a mannose-binding protein-trimannoside complex using residual dipolar couplings

The ligand-binding properties of a 53 kDa homomultimeric trimer from mannose-binding protein (MBP) have been investigated using residual dipolar couplings (RDCs) that are easily measured from NMR spectra of the ligand and isotopically labeled protein. Using a limited set of 1H-15N backbone amide NMR assignments for MBP and orientational information derived from the RDC measurements in aligned media, an order tensor for MBP has been determined that is consistent with symmetry-based predictions of an axially symmetric system. 13C-1H couplings for a bound trisaccharide ligand, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (trimannoside) have been determined at natural abundance and used as orientational constraints. The bound ligand geometry and orientational constraints allowed docking of the trimannoside ligand in the binding site of MBP to produce a structural model for MBP-oligosaccharide interactions.     

NMR identification of left-handed polyproline type II helices

NMR characteristics of a model left-handed 3(1)-helical peptide are reported in this study. With temperature and sequence corrections on the predicted random coil (15)N chemical shifts, a significant (15)N chemical shift deviation is observed for the model 3(1) peptide. The (15)N chemical shift differences also correlate well with the molar ellipticities (at 220 nm) of the CD spectra at different temperatures, indicating that the (15)N chemical shift is a sensitive probe for 3(1)-helices. The average (3)J(HNalpha) and (1)J(CalphaHalpha) values of the model peptide are determined to be 6.5 and 142.6 Hz, respectively, which are consistent with the values calculated from the geometry of 3(1)-helices. With careful measurements of amide (15)N chemical shifts and incorporating temperature and sequence effect corrections, the (15)N chemical shifts can be used together with (3)J(HNalpha) and (1)J(CalphaHalpha) to differentiate 3(1)-helices from random coils with high confidence. Based on the observed NMR characteristics, a strategy is developed for probing left-handed 3(1)-helical structures from other secondary structures.