Klaus Zangger

Simplifying Proton NMR Spectra by Instant Homonuclear Broadband Decoupling

However, these techniques have not been widelyused, since they are insensitive and usually complicatedprocessing schemes have to be applied to achieve the desiredhomonuclear decoupling. Recently, the slice-selective broad-band-decoupling during a weak gradient field (also calledZangger–Sterk or ZS method)

Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites

Metallothioneins (MTs) and various other metal binding proteins release metals when exposed to nitric oxide (NO). We investigated the structural consequences of the interaction between MTs and NO by using 1H‐ and 113Cd‐NMR spectroscopy and found that only the three metals from the N‐terminal β‐domain were selectively released whereas the C‐terminal α‐domain remains intact. Since it has been proposed that the β‐domain is responsible for the postulated role of MTs in zinc homeostasis, whereas the tight binding of metals in the α‐domain appears to play a role in heavy metal detoxification, our results suggest a potential regulatory role of NO in zinc distribution. Specifically, we present a mechanism whereby MT counteracts the cytotoxic effects of NO at inflammatory sites.

Rejuvenation of CcdB-Poisoned Gyrase by an Intrinsically Disordered Protein Domain

Toxin-antitoxin modules are small regulatory circuits that ensure survival of bacterial populations under challenging environmental conditions. The ccd toxin-antitoxin module on the F plasmid codes for the toxin CcdB and its antitoxin CcdA. CcdB poisons gyrase while CcdA actively dissociates CcdB:gyrase complexes in a process called rejuvenation. The CcdA:CcdB ratio modulates autorepression of the ccd operon. The mechanisms behind both rejuvenation and regulation of expression are poorly understood. We show that CcdA binds consecutively to two partially overlapping sites on CcdB, which differ in affinity by six orders of magnitude. The first, picomolar affinity interaction triggers a conformational change in CcdB that initiates the dissociation of CcdB:gyrase complexes by an allosteric segmental binding mechanism. The second, micromolar affinity binding event regulates expression of the ccd operon. Both functions of CcdA, rejuvenation and autoregulation, are mechanistically intertwined and depend crucially on the intrinsically disordered nature of the CcdA C-terminal domain.

The Molecular Chaperone Hsp90 Modulates Intermediate Steps of Amyloid Assembly of the Parkinson-related Protein α-Synuclein

α-Synuclein is an intrinsically unstructured protein that binds to membranes, forms fibrils, and is involved in neurodegeneration. We used a reconstituted in vitro system to show that the molecular chaperone Hsp90 influenced α-synuclein vesicle binding and amyloid fibril formation, two processes that are tightly coupled to α-synuclein folding. Binding of Hsp90 to monomeric α-synuclein occurred in the low micromolar range, involving regions of α-synuclein that are critical for vesicle binding and amyloidogenesis. As a consequence, both processes were affected. In the absence of ATP, the accumulation of non-amyloid α-synuclein oligomers prevailed over fibril formation, whereas ATP favored fibril growth. This suggests that Hsp90 modulates the assembly of α-synuclein in an ATP-dependent manner. We propose that Hsp90 affects these folding processes by restricting conformational fluctuations of α-synuclein.

Boosting the Resolution of 1H NMR Spectra by Homonuclear Broadband Decoupling

Broadband homonuclear decoupling of proton spectra, that is, the collapse of all multiplets into singlets, has the potential of boosting the resolution of (1)H NMR spectra. Several methods have been described in the last 40 years to achieve this goal. Most of them can only be applied in the indirect dimension of multi-dimensional NMR spectra or special data processing is necessary to yield decoupled 1D proton spectra. Recently, complete decoupling of proton spectra during acquisition has been introduced; this not only significantly reduced the experimental time to record these spectra, but also removed the need for any sophisticated processing schemes. Here we present an introduction and overview of the techniques and applications of broadband proton-decoupled proton experiments.

The solution structure of ParD, the antidote of the ParDE toxin–antitoxin module, provides the structural basis for DNA and toxin binding

ParD is the antidote of the plasmid‐encoded toxin–antitoxin (TA) system ParD–ParE. These modules rely on differential stabilities of a highly expressed but labile antidote and a stable toxin expressed from one operon. Consequently, loss of the coding plasmid results in loss of the protective antidote and poisoning of the cell. The antidote protein usually also exhibits an autoregulatory function of the operon. In this paper, we present the solution structure of ParD. The repressor activity of ParD is mediated by the N‐terminal half of the protein, which adopts a ribbon‐helix‐helix (RHH) fold. The C‐terminal half of the protein is unstructured in the absence of its cognate binding partner ParE. Based on homology with other RHH proteins, we present a model of the ParD–DNA interaction, with the antiparallel β‐strand being inserted into the major groove of DNA. The fusion of the N‐terminal DNA‐binding RHH motif to the toxin‐binding unstructured C‐terminal domain is discussed in its evolutionary context.

Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the α-domain

Upon storage under aerobic conditions metallothioneins (MTs) form a new species, which is characterized by a molecular mass approximately twice the size of monomeric MT and shifted (113/111)Cd- and (1)H-NMR resonances. The investigation of this oxidative dimerization process by NMR spectroscopy allowed us to structurally characterize this MT species that has been described to occur in vivo and might be synthesized under conditions of oxidative stress. The oxidative dimer was characterized by the formation of an intermolecular cysteine disulphide bond involving the alpha-domain, and a detailed analysis of chemical shift changes and intermolecular nuclear Overhauser effects points towards a disulphide bond involving Cys(36). In contrast to the metal-bridged (non-oxidative) dimerization, the metal-cysteine cluster structures in both MT domains remain intact and no conformational exchange or metal-metal exchange was observed. Also in contrast to the many recently reported oxidative processes which involve the beta-domain cysteine groups and result in the increased dynamics of the bound metal ions in this N-terminal domain, we found no evidence for any increased dynamics in the alpha-domain metals following this oxidation. Therefore these findings provide additional corroboration that metal binding in the C-terminal alpha-domain is rather tight, even under conditions of a changing cellular oxidation potential, compared with the more labile/dynamic nature of the metals in the N-terminal beta-domain cluster under similar conditions.

Structure of allergens and structure based epitope predictions.

The structure determination of major allergens is a prerequisite for analyzing surface exposed areas of the allergen and for mapping conformational epitopes. These may be determined by experimental methods including crystallographic and NMR-based approaches or predicted by computational methods. In this review we summarize the existing structural information on allergens and their classification in protein fold families. The currently available allergen-antibody complexes are described and the experimentally obtained epitopes compared. Furthermore we discuss established methods for linear and conformational epitope mapping, putting special emphasis on a recently developed approach, which uses the structural similarity of proteins in combination with the experimental cross-reactivity data for epitope prediction.

Positioning of micelle-bound peptides by paramagnetic relaxation enhancements.

Many peptides, proteins, and drugs interact with biological membranes, and knowing the mode of binding is essential to understanding their biological functions. To obtain the complete orientation and immersion depth of such a compound, the membrane-mimetic system (micelle) is placed in an aqueous buffer containing the soluble and inert paramagnetic contrast agent Gd(DTPA-BMA). Paramagnetic relaxation enhancements (PREs) of a specific nucleus then depend only on its distance from the surface. The positioning of a structurally characterized compound can be obtained by least-squares fitting of experimental PREs to the micelle center position. This liquid-state NMR approach, which does not rely on isotopic labeling or chemical modification, has been applied to determine the location of the presumed transmembrane region 7 of yeast V-ATPase (TM7) and the membrane-bound antimicrobial peptide CM15 in micelles. TM7 binds in a trans-micelle orientation with the N-terminus being slightly closer to the surface than the C-terminus. CM15 is immersed unexpectedly deep into the micelle with the more hydrophilic side of the helix being closer to the surface than the hydrophobic one.

Dynamics of interdomain and intermolecular interactions in mammalian metallothioneins

The structures of mammalian metallothioneins (MTs), as solved by X-ray crystallography and NMR spectroscopy, all show seven divalent metals bound in two separate domains. The marked differences in metal-mobilities found for the two domains has led to the proposal for a dual role for the two MT metal domains. The tight metal binding in the C-terminal alpha-domain supposedly constitutes the basis for the detoxification of excess heavy metals, while the more labile metals in the N-terminal beta-domain function in the homeostasis of the essential elements zinc and copper. In this overview, we compare the two types of dimers found for MTs and their influence on metal-mobilities. In the presence of excess metal, the N-terminal domain is responsible for the formation of metal-bridged dimers while under aerobic conditions, a specific intermolecular disulfide is formed between the C-terminal domains. Both forms of dimers not only involve different domains for their intermolecular protein interactions, they also exhibit radical differences in the reactive properties of their respective cluster bound metal ions. Since the metal exchange within each domain is also influenced by interdomain interactions, the relative orientation of the domains is also most likely important for MT functions. Thus far, the relative orientation of the two domains could only be obtained from the crystal structure. Here, we present evidence for increased mobility in the linker region as the reason for the lack of interdomain constraints in the solution NMR studies of mammalian MTs.