HI-NESS: a family of genetically encoded DNA labels based on a bacterial nucleoid-associated protein.

The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding.

Two β-Lactamase Variants with Reduced Clavulanic Acid Inhibition Display Different Millisecond Dynamics.

The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer the most inhibitor resistance, K234R, as well as variant G132N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance (NMR) relaxation experiments to probe structural and dynamic properties.

A Two-Armed Probe for In-Cell DEER Measurements on Proteins*.

The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances.

Efficient Encounter Complex Formation and Electron Transfer to Cytochrome c Peroxidase with an Additional, Distant Electrostatic Binding Site.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined.

The Transient Complex of Cytochrome c and Cytochrome c Peroxidase: Insights into the Encounter Complex from Multifrequency EPR and NMR Spectroscopy.

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL-Cc). A dissociation constant K of 20±4×10  M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found.

β-Lactamase of Mycobacterium tuberculosis Shows Dynamics in the Active Site That Increase upon Inhibitor Binding.

The β-lactamase BlaC is a broad-spectrum β-lactamase that can convert a range of β-lactam antibiotics. Enzymes with low specificity are expected to exhibit active-site flexibility. To probe the motions in BlaC, we studied the dynamic behavior in solution using nuclear magnetic resonance (NMR) spectroscopy.

A Double-Armed, Hydrophilic Transition Metal Complex as a Paramagnetic NMR Probe.

Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protein complexes. Herein, two transition metal NMR probes (TraNPs) are reported. TraNPs are attached through two arms to a protein to generate a pseudocontact shift (PCS) using cobalt(II), or paramagnetic relaxation enhancement (PRE) with manganese(II).

New Conformations of Acylation Adducts of Inhibitors of β-Lactamase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to β-lactam antibiotics due to the production of the extended spectrum β-lactamase BlaC. β-Lactam/β-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the β-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam.

Methyl group reorientation under ligand binding probed by pseudocontact shifts.

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand.

Methyl group assignment using pseudocontact shifts with PARAssign.

A new version of the program PARAssign has been evaluated for assignment of NMR resonances of the 76 methyl groups in leucines, isoleucines and valines in a 25 kDa protein, using only the structure of the protein and pseudocontact shifts (PCS) generated with a lanthanoid tag at up to three attachment sites. The number of reliable assignments depends strongly on two factors. The principle axes of the magnetic susceptibility tensors of the paramagnetic centers should not be parallel so as to avoid correlated PCS.

Phosphate Promotes the Recovery of Mycobacterium tuberculosis β-Lactamase from Clavulanic Acid Inhibition.

The rise of multi- and even totally antibiotic resistant forms of Mycobacterium tuberculosis underlines the need for new antibiotics. The pathogen is resistant to β-lactam compounds due to its native serine β-lactamase, BlaC. This resistance can be circumvented by administration of a β-lactamase inhibitor.

Delicate conformational balance of the redox enzyme cytochrome P450cam.

The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution.

A two-armed lanthanoid-chelating paramagnetic NMR probe linked to proteins via thioether linkages.

Paramagnetic NMR probes provide valuable long-range structural information on proteins and protein complexes. A new, stable, two-armed lanthanoid probe is reported that can be attached to a protein site-specifically via chemically inert thioether linkages.

An ensemble of rapidly interconverting orientations in electrostatic protein-peptide complexes characterized by NMR spectroscopy.

Protein complex formation involves an encounter state in which the proteins are associated in a nonspecific manner and often stabilized by interactions between charged surface patches. Such patches are thought to bind in many different orientations with similar affinity. To obtain experimental evidence for the dynamics in encounter complexes, a model was created using the electron transfer protein plastocyanin and short charged peptides.

The structure of the cytochrome p450cam-putidaredoxin complex determined by paramagnetic NMR spectroscopy and crystallography.

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system.

A pH-sensitive, colorful, lanthanide-chelating paramagnetic NMR probe.

Paramagnetic lanthanides ions are broadly used in NMR spectroscopy. The effects of unpaired electrons on NMR spectral parameters provide a powerful tool for the characterization of macromolecular structures and dynamics. Here, a new lanthanide-chelating NMR probe, Caged Lanthanide NMR Probe-7 (CLaNP-7), is presented.

Efficient electron transfer in a protein network lacking specific interactions.

In many biochemical processes, proteins need to bind partners amidst a sea of other molecules. Generally, partner selection is achieved by formation of a single-orientation complex with well-defined, short-range interactions. We describe a protein network that functions effectively in a metabolic electron transfer process but lacks such specific interactions.

Amicyanin transfers electrons from methylamine dehydrogenase to cytochrome c-551i via a ping-pong mechanism, not a ternary complex.

The first crystal structure of a ternary redox protein complex was comprised of the enzyme methylamine dehydrogenase (MADH) and two electron transfer proteins, amicyanin and cytochrome c-551i from Paracoccus denitrificans [Chen et al. Science 1994, 264, 86-90]. The arrangement of the proteins suggested possible electron transfer from the active site of MADH via the amicyanin copper ion to the cytochrome heme iron, although the distance between the metals is large.

Validation of a lanthanide tag for the analysis of protein dynamics by paramagnetic NMR spectroscopy.

Paramagnetic lanthanide tags potentially can enhance the effects of microsecond to millisecond dynamics in proteins on NMR signals and provide structural information on lowly populated states encoded in the pseudocontact shifts. We have investigated the microsecond to millisecond mobility of a two-point attached lanthanide tag, CLaNP-5, using paramagnetic (1)H CPMG relaxation dispersion methods. CLaNP-5 loaded with Lu(3+), Yb(3+), or Tm(3+) was attached to three sites on the surface of two proteins, pseudoazurin and cytochrome c.

A Proton ENDOR Study of Azurin.

As part of our ongoing project that aims at the optimum characterization of the electronic structure of the blue-copper site of azurin from Pseudomonas aeruginosa, we present the complete hyperfine tensors of the protons bound to the Cbeta atom of the copper-bound cysteine 112. These tensors have been obtained from a 95 GHz pulsed electron-nuclear double resonance study of a single crystal of the protein.

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.