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Ahmad, M. U. D. et al. Nano-differential scanning fluorimetry for screening in fragment-based lead discovery. Journal of Visualized Experiments: JoVE. (171), e62469 (2021). Barbieri, L., Luchinat, E. Monitoring protein-ligand interactions in human cells by real-time quantitative in-cell NMR using a high cell density bioreactor. Journal of Visualized Experiments: JoVE. (169), e62323 (2021). Berg, H. et al. NMR-based fragment screening in a minimum sample but maximum automation mode. Journal of Visualized Experiments: JoVE. (172), e62262 (2021). Beriashvili, D., Schellevis, R. D., Napoli, F., Weingarth, M., Baldus, M. High-resolution studies of proteins in natural membranes by solid-state NMR. Journal of Visualized Experiments: JoVE. (169), e62197 (2021). Cornaciu, I. et al. The automated crystallography pipelines at the EMBL HTX facility in grenoble. Journal of Visualized Experiments: JoVE. (172), e62200 (2021). Crawshaw, A. et al. A sample preparation pipeline for microcrystals at the VMXm beamline. Journal of Visualized Experiments: JoVE. (172), e62306 (2021). Douangamath, A. et al. Achieving efficient fragment screening at XChem facility at diamond light source. Journal of Visualized Experiments: JoVE. (171), e62414 (2021). Groen, J. et al. A 3D cartographic description of the cell by Cryo Soft X-ray tomography. Journal of Visualized Experiments: JoVE. (169), e62190 (2021). Horrell, S. et al. Fixed target serial data collection at diamond light source. Journal of Visualized Experiments: JoVE. (168), e62491 (2021). Jiménez-Moreno, A. et al. Cryo-EM and single-particle analysis with scipion. Journal of Visualized Experiments: JoVE. (171), e62261 (2021). Moravcová, J., Pinkas, M., Holbová, R., Nováček, J. Preparation and CryoFIB micromachining of Saccharomyces cerevisiae for CryoElectron tomography. Journal of Visualized Experiments: JoVE. e62351 (2021). Vyas, N. et al. Cryo-structured illumination microscopic data collection from cryogenically preserved cells. Journal of Visualized Experiments: JoVE. (171), e62274 (2021). Weis, F., Hagen, W. J. H., Schorb, M., Mattei, S. Strategies for optimization of cryogenic electron tomography data acquisition. Journal of Visualized Experiments: JoVE. (169), e62383 (2021). White, J. B. R. et al. Single particle cryo-electron microscopy: From sample to structure. Journal of Visualized Experiments: JoVE. (171), e62415 (2021). Wollenhaupt, J. et al. Workflow and tools for crystallographic fragment screening at the helmholtz-zentrum Berlin. Journal of Visualized Experiments: JoVE. (169), e62208 (2021).

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

iNEXT, ‘Infrastructure for NMR, EM and X‐rays for Translational research’, is a EC‐funded Horizon 2020 network of national infrastructures aiming to facilitate EC‐supported, trans‐national user access to several high‐end structural biology infrastructures and to expand the use of structural biology technologies towards new research communities. iNEXT has developed different access routes offering cutting edge technologies and expertise, thus targeting the diverse needs of research groups that desire to perform X‐ray crystallography, small‐angle X‐ray scattering, solution or solid‐state NMR, single particle or cell tomography electron microscopy, biophysics, and imaging experiments. Specific Joint Research Activities aim at improving the user experience in ever advancing methods, and are focused on structure‐based drug discovery, membrane protein characterization and structural cell biology initiatives. iNEXT Consortium members Rolf Boelens (Coordinator): Universiteit Utrecht, Utrecht, The Netherlands; Dmitri Svergun, Jan Ellenberg, Stephen Cusack: European Molecular Biology Laboratory (Hamburg, Heidelberg and Grenoble sites, respectively); Martin A. Walsh, Diamond Light Source Limited, Didcot, United Kingdom; Lucia Banci: Consorzio Interuniversitario Risonanze Magnetiche di Metallo Proteine, Firenze, Italy; Harald Schwalbe, Johann Wolfgang Goethe Universitaet, Frankfurt am Main, Germany; Anastassis Perrakis: Stichting het Nederlands Kanker Instituut ‐ Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, The Netherlands; Vladimír Sklenář, Masarykova Univerzita, Brno, Czech Republic; Jose Maria Carazo Garcia: Agencia Estatal Consejo Superior de Investigaciones Cientificas, Madrid, Spain; Hartmut Oschkinat: Forschungsverbund Berlin E.V., Berlin, Germany; Andrew Thompson: Société Civile Synchrotron SOLEIL, Gif sur Yvette, France; Marjolein Thunnissen: Lunds Universitet, Lund, Sweden; Bram Koster: Universiteit Leiden and Leids Universitair Medisch Centrum, Leiden, The Netherlands; Gordon Leonard: Installation Europeenne de Rayonnement Synchrotron, Grenoble, France; Bernhard Brutscher: Centre National de la Recherche Scientifique, Grenoble/Lyon, France; David I. Stuart: INSTRUCT Academic Services Limited, Oxford, United Kingdom; Poul Nissen: Aarhus Universitet, Aarhus, Denmark; Hanna Wacklin: European Spallation Source ESS AB, Lund, Sweden; András Perczel: Eotvos Lorand Tudomanyegyetem, Budapest, Hungary; Margarida Archer: Instituto de Tecnologia Quimica e Biologica, Oeiras, Portugal; Rik Wierenga: Oulun Yliopisto, Oulu, Finland; Georgios A. Spyroulias: University of Patras, Rio Patras, Greece; Joel Sussman: Weizmann Institute of Science, Rehovot, Israel.

Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2-30S interaction, is positioned between the GTP-binding G2 and the fMet-tRNA binding C-terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β-sheets with each four anti-parallel strands, followed by a C-terminal α-helix. In line with its role as linker between G3 and subdomain C1, this helix has no well-defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB-fold module, similar to that of the corresponding subdomain of Thermus thermophilus IF2, and to that of other known RNA-binding modules such as IF2-C2, IF1 and subdomains II of elongation factors EF-Tu and EF-G. Structural comparisons have resulted in a model that describes the interaction between IF2-G3 and the 30S ribosomal subunit.

Signalling by cyclic adenosine monophosphate (cAMP) occurs via various effector proteins, notably protein kinase A and the guanine nucleotide exchange factors Epac1 and Epac2. These proteins are activated by cAMP binding to conserved cyclic nucleotide binding domains. The specific roles of the effector proteins in various processes in different types of cells are still not well defined, but investigations have been facilitated by the development of cyclic nucleotide analogues with distinct selectivity profiles towards a single effector protein. A remaining challenge in the development of such analogues is the poor membrane permeability of nucleotides, which limits their applicability in intact living cells. Here, we report the synthesis and characterisation of S223-AM, a cAMP analogue designed as an acetoxymethyl ester prodrug to overcome limitations of permeability. Using total internal reflection imaging with various fluorescent reporters, we show that S223-AM selectively activates Epac2, but not Epac1 or protein kinase A, in intact insulin-secreting &beta, cells, and that this effect was associated with pronounced activation of the small G-protein Rap. A comparison of the effects of different cAMP analogues in pancreatic islet cells deficient in Epac1 and Epac2 demonstrates that cAMP-dependent Rap activity at the &beta, cell plasma membrane is exclusively dependent on Epac2. With its excellent selectivity and permeability properties, S223-AM should get broad utility in investigations of cAMP effector involvement in many different types of cells.

Genome replication, transcription and repair require the assembly/disassembly of the nucleosome. Histone chaperones are regulators of this process by preventing formation of non-nucleosomal histone–DNA complexes. Aprataxin and polynucleotide kinase like factor (APLF) is a non-homologous end-joining (NHEJ) DNA repair factor that possesses histone chaperone activity in its acidic domain (APLFAD). Here, we studied the molecular basis of this activity using biochemical and structural methods. We find that APLFAD is intrinsically disordered and binds histone complexes (H3-H4)2 and H2A-H2B specifically and with high affinity. APLFAD prevents unspecific complex formation between H2A-H2B and DNA in a chaperone assay, establishing for the first time its specific histone chaperone function for H2A-H2B. On the basis of a series of nuclear magnetic resonance studies, supported by mutational analysis, we show that the APLFAD histone binding domain uses two aromatic side chains to anchor to the α1–α2 patches on both H2A and H2B, thereby covering most of their DNA-interaction surface. An additional binding site on both APLFAD and H2A-H2B may be involved in the handoff between APLF and DNA or other chaperones. Together, our data support the view that APLF provides not only a scaffold but also generic histone chaperone activity for the NHEJ-complex.

Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D-structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N-terminal region, and four out of six cysteine pairs have been assigned by 2D-NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat-denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein keeps its native 3D-structure up to 90°C, but it becomes unfolded at temperatures of 100-120°C. Heat denaturation affects the immunological properties of both peach allergens, which lose at least partially their IgE-binding epitopes. In conclusion, the data collected in this study provide a first set of information on the molecular properties of Peamaclein. Future studies could lead to the possible use of the denatured form of this protein as a vaccine, and of the inclusion of cooked peach in the diet of subjects allergic to Peamaclein.

Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.

Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D-structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N-terminal region, and four out of six cysteine pairs have been assigned by 2D-NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat-denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein keeps its native 3D-structure up to 90°C, but it becomes unfolded at temperatures of 100-120°C. Heat denaturation affects the immunological properties of both peach allergens, which lose at least partially their IgE-binding epitopes. In conclusion, the data collected in this study provide a first set of information on the molecular properties of Peamaclein. Future studies could lead to the possible use of the denatured form of this protein as a vaccine, and of the inclusion of cooked peach in the diet of subjects allergic to Peamaclein. 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 416-425, 2014.

Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet‐tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2‐30S interaction, is positioned between the GTP‐binding G2 and the fMet‐tRNA binding C‐terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β‐sheets with each four anti‐parallel strands, followed by a C‐terminal α‐helix. In line with its role as linker between G3 and subdomain C1, this helix has no well‐defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB‐fold module, similar to that of the corresponding subdomain of Thermus thermophilus IF2, and to that of other known RNA‐binding modules such as IF2‐C2, IF1 and subdomains II of elongation factors EF‐Tu and EF‐G. Structural comparisons have resulted in a model that describes the interaction between IF2‐G3 and the 30S ribosomal subunit.

Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B-Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7-Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.

Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-pi interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 "methyl-methyl" switch in the histone code dynamically regulates TFIID-promoter association.

Sialic acid supports nerve cell regeneration, differentiation and neuronal plasticity. Especially, polysialic acid (polySia) chains which are built up by α2,8-linked Neu5Ac Neu5Ac residues influence by their specific interactions with polySia receptors neuronal processes related to tumor spread and differentiation processes. With a combination of biophysical and biochemical methods including molecular modeling as described here it is possible to support cell biological experiments and in vivo studies on a nanoscale level. The submolecular analytical approaches which are directed to crucial functional groups focus on the potential therapeutic impact of sialic acids and in particular polySia. Such results are helpful for the development of new drugs which might have a high clinical relevance in respect to the therapy of various diseases correlated to neuronal regeneration, tumor spread and infections. It is not surprising that several diseases belonging to these different clinical fields (e.g. oncology, infection diseases, neuronal disorder) can be treated as indicated because sialic acids represent essential contact structures on numerous cell surfaces in dependence to their state of differentiation.

Xylanase from Bacillus agaradhaerens belongs to a large group of glycosyl hydrolases which catalyze the degradation of xylan. The protonation behavior of titratable groups of the uniformly (15)N- and (13)C-labeled xylanase was investigated by multinuclear NMR spectroscopy. A total of 224 chemical shift titration curves corresponding to (1)H, (13)C, and (15)N resonances revealed pK(a) values for all aspartic and glutamic acid residues, as well as for the C-terminal carboxylate and histidine residues. Most of the titratable groups exhibit a complex titration behavior, which is most likely due to the mutual interactions with other neighboring groups or due to an unusual local microenvironment. Subsite -1 containing the catalytic dyad shows a long-range interaction over 9 A with Asp21 via two hydrogen bonds with Asn45 as the mediator. This result illuminates the pivotal role of the conserved position 45 among family 11 endoxylanases, determining an alkaline pH optimum by asparagine residues or an acidic pH optimum by an aspartate. The asymmetric interactions of neighboring tryptophan side chains with respect to the catalytic dyad can be comprehended as a result of hydrogen bonding and aromatic stacking. Most of the chemical shift-pH profiles of the backbone amides exhibit biphasic behavior with two distinct inflection points, which correspond to the pK(a) values of the nearby acidic side chains. However, the alternation of both positive and negative slopes of individual amide titration curves is interpreted as a consequence of a simultaneous reorganization of side chain conformational space at pH approximately 6 and/or an overall change in the hydrogen network in the substrate binding cleft.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the λ-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined α-helices (residues 5–11 and 18–27) and five β-strands (residues 2–4, 30–31, 35–36, 41–44 and 48–49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage λ excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical ‘winged-helix’ DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51–72, which were not resolved in the λ Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.

The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes. Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner. In this study, we investigated the molecular interaction between two components of the electron-transfer chain from Paracoccus denitrificans: the engineered, water-soluble fragment of cytochrome c(552) and the Cu(A) domain from the cytochrome c oxidase. Comparison of [(15)N,(1)H]-TROSY spectra of the [(15)N]-labeled cytochrome c(552) fragment in the absence and in the presence of the Cu(A) fragment showed chemical shift changes for the backbone amide groups of several, mostly uncharged residues located around the exposed heme edge in cytochrome c(552). The detected contact areas on the cytochrome c(552) surface were comparable under both fully reduced and fully oxidized conditions, suggesting that the respective chemical shift changes represent biologically relevant protein-protein interactions.

A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone (1)J(NC'), (1)J(NCalpha), and (2)J(NCalpha) coupling constants are obtained for the two [(15)N;(13)C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [(2)H;(15)N;(13)C]-labeled, large protein DFPase. A dependence of one-bond and two-bond J(NCalpha) values on protein backbone psi torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated (15)N-(13)C residual dipolar couplings.

Bromodomains are epigenetic reader domains, which have come under increasing scrutiny both from academic and pharmaceutical research groups. Effective targeting of the BAZ2B bromodomain by small molecule inhibitors has been recently reported, but no structural information is yet available on the interaction with its natural binding partner, acetylated histone H3K14ac. We have assigned the BAZ2B bromodomain and studied its interaction with H3K14ac acetylated peptides by NMR spectroscopy using both chemical shift perturbation (CSP) data and clean chemical exchange (CLEANEX-PM) NMR experiments. The latter was used to characterize water molecules known to play an important role in mediating interactions. Besides the anticipated Kac binding site, we consistently found the bromodomain BC loop as hotspots for the interaction. This information was used to create a data-driven model for the complex using HADDOCK. Our findings provide both structure and dynamics characterization that will be useful in the quest for potent and selective inhibitors to probe the function of the BAZ2B bromodomain.

The ability of preferredoxin to inactivate a 50-pS anion channel of the chloroplast inner membrane in the presence of an energy source was investigated using single-channel recordings. It was found that preferredoxin cannot inactivate the channel when GTP is the only energy source present. From this it is concluded that the precursor has to interact with the , translocon of the inner membrane of chloroplasts (Tic) complex to be able to inactivate the 50-pS anion channel. The ability of two mutants of preferredoxin with deletions in their transit sequence to inactivate the channel was also tested. Both mutants have been shown to have a similar binding affinity for the chloroplast envelope, but only one is able to fully translocate. The mutants were both able to inactivate the channel in a similar manner. From this it is concluded that full translocation is not necessary for the inactivation of the channel. It is also shown that preferredoxin is capable of inactivating the 50-pS anion channel in the chloroplast-attached configuration as was previously found in the inside-out configuration. From this it is concluded that stromal factors do not influence the protein-import-induced inactivation of the 50-pS anion channel of the chloroplast inner membrane. Finally the effect of the anion channel blocker 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) on the channel activity and on protein import was investigated. It was found that DIDS blocked the channel. Furthermore the addition of the channel blocker reduces the efficiency of import to 52%. This leads to the conclusion that correct functioning of the channel is important for protein import.

For the first time a method is described in which an N-terminal targeting peptide is isolated from Escherichia coli. After overexpression, purification, and cleavage of a fusion protein the protease-sensitive transit peptide from the chloroplast precursor protein preferredoxin could be isolated by HPLC. It was characterized by N-terminal amino acid sequencing and electrospray mass spectrometry. Its functionality was suggested by in vitro import competition experiments with isolated pea chloroplasts, in which the isolated peptide inhibited the import of radioactively labeled preferredoxin. Results from import competition experiments performed with a transit peptide deletion mutant suggested that the four extreme C-terminal amino acids lack information to interact with the chloroplast import machinery.

Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.

In order to analyze the information content of a chloroplast transit sequence, we have constructed and analyzed by in vitro assays seven substitution and 20 deletion mutants of the ferredoxin transit sequence. The N-terminal part and the C-terminal part are important for targeting, and in addition the C-terminal region is required for processing. A third region is important for translocation but not for the initial interaction with the envelope. A fourth region is less essential for in vitro import. Purified precursors were tested for their ability to compete for the in vitro import of radiolabeled wild-type precursor, which confirmed the important role in chloroplast recognition of both the N- and the C-terminal domain of the transit sequence. Monolayer experiments showed that the N terminus was mainly involved in the insertion into mono-galactolipid-containing lipid surfaces whereas the C terminus mediates the recognition of negatively charged lipids. A sequence comparison to other transit sequences suggests that the domain structure of the ferredoxin transit sequence can be extended to these sequences and thus reveals a general structural design of transit sequences.

Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The resulting mutant retains significant catalytic activity upon heating above 60 °C (and up to 100 °C) and cooling down, whereas wild-type lipase precipitates irreversibly and does not show significant activity in these conditions. Kinetic thermostability of proteins has not been characterized well on the molecular structure level so far, therefore our aim is to study it using NMR spectroscopy. Here, nearly complete (1)H, (13)C and (15)N resonance assignments are reported for wild-type and mutant Lipase A. Chemical shifts were used to predict secondary structure elements of both Lipase A variants.

Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acanthaporin, to our knowledge the first pore-forming toxin to be described from acanthamoebae, which are free-living, bacteria-feeding, unicellular organisms that are opportunistic pathogens of increasing importance and cause severe and often fatal diseases. We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by tracking its pore-forming activity, molecularly cloned the gene of its precursor and recombinantly expressed the mature protein in bacteria. Acanthaporin was cytotoxic for human neuronal cells and exerted antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes. The tertiary structures of acanthaporin's active monomeric form and inactive dimeric form, both solved by NMR spectroscopy, revealed a currently unknown protein fold and a pH-dependent trigger mechanism of activation.

Escherichia coli strain AD93 is unable to synthesize the nonbilayer lipid phosphatidylethanolamine and requires high concentrations of specific divalent cations for growth. Previous studies suggested that in this strain, cardiolipin in combination with divalent cations functionally replaces phosphatidylethanolamine, reflecting polymorphic regulation of membrane lipid composition. However, it is also possible that divalent cations are required for regulation of lipid packing or membrane surface potential. 2H NMR was employed to measure the effect of different divalent cations on lipid packing in aqueous dispersions of lipid extracts isolated from AD93 and the wild type parental strain W3899, which were grown with [11,11-2H2]oleic acid. The results indicate that a range of acyl chain order is compatible with growth and that Ba2+, which cannot support growth of AD93, can increase chain packing to the wild type level. By means of microelectrophoresis, it was shown that the growth-promoting cations and Ba2+ have a strong and comparable ability to screen the surface charge of large unilamellar vesicles prepared from AD93 lipid extracts. Therefore, it is unlikely that the growth-promoting capacity of divalent cations is primarily due to their effect on lipid packing or their potency to decrease the surface potential. Furthermore, the addition of small amounts of Ba2+ to a AD93 lipid dispersion with excess Mg2+ diminished HII phase formation. This observation can explain the growth arrest in AD93 cultures upon the addition of Ba2+ and further supports the conclusion that the cation requirement of this strain arises mainly from polymorphic regulation of lipid composition.

Binding of the nuclear factor-Y complex (NF-Y) to the inverted CCAAT-box interferes with transcription activation through nucleosome reorganization. The three homologous proteins forming the zinc-fingers and homeoboxes (ZHX) family interact with the activation domain of NF-Ya to repress transcription. Each ZHX-protein contains two generic C2H2 zinc-fingers (ZNF1 and ZNF2) followed by five homeodomains. Although the proteins have been related to the occurrence of certain cancers, the function and structure of the individual ZHX domains are still unknown. Here, we determined the structure of the tandem zinc-finger region of human ZHX1. Folding and secondary structure predictions combined with expression screening revealed that the C-terminal extension (E) to ZNF2 could form a single domain with the two hZHX1 zinc-fingers. We therefore decided to determine the solution structure of the zinc-fingers followed by this extension. We show that both zinc-fingers adopt canonical betabetaalpha-folds in which a zinc ion is coordinated by two cysteine and two histidine residues. The C-terminal extension to ZNF2 forms two beta-strands to make a beta-sheet with the beta-strands of this zinc-finger. The ZNF1 and ZNF2-E domains do not show evident contacts and their mutual orientation seems variable. The high degree of sequence conservation among ZHX family members permitted us to prepare homology models for ZHX2 and ZHX3, revealing distinct surface characteristics for each family member. Implications of these structural features for ZHX-functioning in transcription regulation are discussed.

Rapid progress has been made in the field of protein design over the past couple of years. Computational design algorithms have been used to (i) stabilize naturally occurring proteins,[1][2] (ii) increase the solubility of membrane proteins,[3] and (iii) create de novo proteins that feature previously unknown structural folds.[4] Hence, computational protein design opens a multitude of possibilities to study the complex processes of protein folding and to possibly create new structures with novel functional properties. The first completely de novo designed protein TOP7 (PDB 1QYS), for example, provided the opportunity to explore the energy landscape of a protein structure that was never exposed to natural selection pressure.[5] The long-term goal of this research field is the ability to design proteins that perform the desired functions and exhibit high stability for possible application in industry and therapy.

Epac1 is a cAMP-responsive exchange factor for the small G-protein Rap. It consists of a regulatory region containing a cyclic nucleotide binding (CNB) domain and a catalytic region that activates Rap. In the absence of cAMP, access of Rap to the catalytic site is blocked by the regulatory region. We analyzed the conformational states of the CNB domain in the absence and in the presence of cAMP and cAMP analogues by NMR spectroscopy, resulting in the first direct insights into the activation mechanism of Epac. We prove that the CNB domain exists in equilibrium between the inactive and the active conformation, which is shifted by binding of cAMP. cAMP binding results in conformational changes in both the ligand binding pocket and the outer helical segments. We used two different cAMP antagonists that block these successive changes to elucidate the steps of this process. Highlighting the role of dynamics, the superactivator 8-pCPT-2'-O-Me-cAMP induces similar conformational changes as cAMP but causes different internal mobility. The results reveal the critical elements of the CNB domain of Epac required for activation and highlight the role of dynamics in this process.

IF2 is one of three bacterial translation initiation factors that are conserved through all kingdoms of life. It binds the 30S and 50S ribosomal subunits, as well as fMet-tRNAf(Met). After these interactions, fMet-tRNAf(Met) is oriented to the ribosomal P-site where the first amino acid of the nascent polypeptide, formylmethionine, is presented. The C-terminal domain of Bacillus stearothermophilus IF2, which is responsible for recognition and binding of fMet-tRNAf(Met), contains two structured modules. Previously, the solution structure of the most C-terminal module, IF2-C2, has been elucidated by NMR spectroscopy and direct interactions between this subdomain and fMet-tRNAf(Met) were reported. In the present NMR study we have obtained the spectral assignment of the other module of the C-terminal domain (IF2-C1) and determined its solution structure and backbone dynamics. The IF2-C1 core forms a flattened fold consisting of a central four-stranded parallel beta-sheet flanked by three alpha-helices. Although its overall organization resembles that of subdomain III of the archaeal IF2-homolog eIF5B whose crystal structure had previously been reported, some differences of potential functional significance are evident.

The periplasmic polysulfide-sulfur transferase (Sud) protein encoded by Wolinella succinogenes is involved in oxidative phosphorylation with polysulfide-sulfur as a terminal electron acceptor. The polysulfide-sulfur is covalently bound to the catalytic Cys residue of the Sud protein and transferred to the active site of the membranous polysulfide reductase. The solution structure of the homodimeric Sud protein has been determined using heteronuclear multidimensional NMR techniques. The structure is based on NOE-derived distance restraints, backbone hydrogen bonds, and torsion angle restraints as well as residual dipolar coupling restraints for a refinement of the relative orientation of the monomer units. The monomer structure consists of a five-stranded parallel beta-sheet enclosing a hydrophobic core, a two-stranded antiparallel beta-sheet, and six alpha-helices. The dimer fold is stabilized by hydrophobic residues and ion pairs found in the contact area between the two monomers. Similar to rhodanese enzymes, Sud catalyzes the transfer of the polysulfide-sulfur to the artificial acceptor cyanide. Despite their similar functions and active sites, the amino acid sequences and structures of these proteins are quite different.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the lambda-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28--Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined alpha-helices (residues 5-11 and 18-27) and five beta-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage lambda excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical 'winged-helix' DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51-72, which were not resolved in the lambda Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.

Nuclear-encoded, chloroplast-destined proteins are synthesized with transit sequences that contain all information to get them inside the organelle. Different proteins are imported via a general protein import machinery, but their transit sequences do not share amino acid homology. It has been suggested that interactions between transit sequence and chloroplast envelope membrane lipids give rise to recognizable, structural motifs. In this study a detailed investigation of the structural, dynamical, and topological features of an isolated transit peptide associated with mixed micelles is described. The structure of the preferredoxin transit peptide in these micelles was studied by circular dichroism (CD) and multidimensional NMR techniques. CD experiments indicated that the peptide, which is unstructured in aqueous solution, obtained helical structure in the presence of the micelles. By NMR it is shown that the micelles introduced ill-defined helical structures in the transit peptide. Heteronuclear relaxation experiments showed that the whole peptide backbone is very flexible. The least dynamic segments are two N- and C-terminal helical regions flanking an unstructured proline-rich amino acid stretch. Finally, the insertion of the peptide backbone in the hydrophobic interior of the micelle was investigated by use of hydrophobic spin-labels. The combined data result in a model of the transit peptide structure, backbone dynamics, and insertion upon its interaction with mixed micelles.

In order to obtain insight into the structural flexibility of chloroplast targeting sequences, the Silene pratensis preferredoxin transit peptide was studied by circular dichroism and nuclear magnetic resonance spectroscopy. In water, the peptide is unstructured, with a minor propensity towards helix formation from Val-9 to Ser-12 and from Gly-30 to Ser-40. In 50% (v/v) trifluoroethanol, structurally independent N- and C-terminal helices are stabilized. The N-terminal helix appears to be amphipathic, with hydrophobic and hydroxylated amino acids on opposite sides. The C-terminal helix comprises amino acids Met-29–Gly-50 and is destabilized at Gly-39. No ordered tertiary structure was observed. The results are discussed in terms of protein import into chloroplasts, in which the possible interactions between the transit peptide and lipids are emphasized.

The exopolysaccharide produced by Lactobacillus paracasei 34-1 in a semi-defined medium was found to be a heteropolymer, composed of d -galactose, 2-acetamido-2-deoxy- d -galactose, and sn -glycerol 3-phosphate in molar ratios of 3:1:1. By means of deglycerophosphorylation, methylation analysis, and 1D/2D NMR studies ( 1 H, 13 C, and 31 P) the polysaccharide was shown to consist of repeating units with the following structure