In situ captured antibacterial action of membrane-incising peptide lamellae.

Developing unique mechanisms of action are essential to combat the growing issue of antimicrobial resistance. Supramolecular assemblies combining the improved biostability of non-natural compounds with the complex membrane-attacking mechanisms of natural peptides are promising alternatives to conventional antibiotics. However, for such compounds the direct visual insight on antibacterial action is still lacking. Here we employ a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular structures of lysine-rich heterochiral ß3-peptides, termed lamellin-2K and lamellin-3K, triggered by bacterial cell surface lipopolysaccharides. Combined molecular dynamics simulations, EM and bacterial assays confirmed that the phosphate-induced conformational change on these lamellins led to the formation of striped lamellae capable of incising the cell envelope of Gram-negative bacteria thereby exerting antibacterial activity. Our findings also provide a mechanistic link for membrane-targeting agents depicting the antibiotic mechanism derived from the in-situ formation of active supramolecules.

A GFP Inspired 8-Methoxyquinoline-Derived Fluorescent Molecular Sensor for the Detection of Zn2+ by Two-Photon Microscopy.

An effective, GFP-inspired fluorescent Zn2+ sensor is developed for two-photon microscopy and related biological application that features an 8-methoxyquinoline moiety. Excellent photophysical characteristics including a 37-fold fluorescence enhancement with excitation and emission maxima at 440 nm and 505 nm, respectively, as well as a high two-photon cross-section of 73 GM at 880 nm are reported. Based on the experimental data, the relationship between the structure and properties was elucidated and explained backed up by DFT calculations, particularly the observed PeT phenomenon for the turn-on process. Biological validation and detailed experimental and theoretical characterization of the free and the zinc-bound compounds are presented.

Assignment of the disordered, proline-rich N-terminal domain of the tumour suppressor p53 protein using 1HN and 1Hα-detected NMR measurements.

Protein p53 is mostly known for playing a key role in tumour suppression, and mutations in the p53 gene are amongst the most frequent genomic events accompanying oncogenic transformation. Continuous research is conducted to target disordered proteins/protein regions for cancer therapy, for which atomic level information is also necessary. The disordered N-terminal part of p53 contains the transactivation and the proline-rich domains-which besides being abundant in proline residues-contains repetitive Pro-Ala motifs. NMR assignment of such repetitive, proline-rich regions is challenging due to the lack of amide protons in the 1HN-detected approaches, as well as due to the small chemical shift dispersion. In the present study we perform the full assignment of the p531-100 region by applying a combination of 1HN- and 1Hα-detected NMR experiments. We also show the increased information content when using real-time homo- and heteronuclear decoupled acquisition schemes. On the other hand, we highlight the presence of minor proline species, and using Pro-selective experiments we determine the corresponding cis or trans conformation. Secondary chemical shifts for (Cα-Cß) atoms indicate the disordered nature of this region, with expected helical tendency for the TAD1 region. As the role of the proline-rich domain is yet not well understood our results can contribute to further successful investigations.

Proline cis/trans Isomerization in Intrinsically Disordered Proteins and Peptides.

BACKGROUND: Intrinsically disordered proteins and protein regions (IDPs/IDRs) are important in diverse biological processes. Lacking a stable secondary structure, they display an ensemble of conformations. One factor contributing to this conformational heterogeneity is the proline cis/trans isomerization. The knowledge and value of a given cis/trans proline ratio are paramount, as the different conformational states can be responsible for different biological functions. Nuclear Magnetic Resonance (NMR) spectroscopy is the only method to characterize the two co-existing isomers on an atomic level, and only a few works report on these data. METHODS: After collecting the available experimental literature findings, we conducted a statistical analysis regarding the influence of the neighboring amino acid types (i ± 4 regions) on forming a cis-Pro isomer. Based on this, several regularities were formulated. NMR spectroscopy was then used to define the cis-Pro content on model peptides and desired point mutations. RESULTS: Analysis of NMR spectra prove the dependence of the cis-Pro content on the type of the neighboring amino acid-with special attention on aromatic and positively charged sidechains. CONCLUSIONS: Our results may benefit the design of protein regions with a given cis-Pro content, and contribute to a better understanding of the roles and functions of IDPs.

The influence of random-coil chemical shifts on the assessment of structural propensities in folded proteins and IDPs.

In studying secondary structural propensities of proteins by nuclear magnetic resonance (NMR) spectroscopy, secondary chemical shifts (SCSs) serve as the primary atomic scale observables. For SCS calculation, the selection of an appropriate random coil chemical shift (RCCS) dataset is a crucial step, especially when investigating intrinsically disordered proteins (IDPs). The scientific literature is abundant in such datasets, however, the effect of choosing one over all the others in a concrete application has not yet been studied thoroughly and systematically. Hereby, we review the available RCCS prediction methods and to compare them, we conduct statistical inference by means of the nonparametric sum of ranking differences and comparison of ranks to random numbers (SRD-CRRN) method. We try to find the RCCS predictors best representing the general consensus regarding secondary structural propensities. The existence and the magnitude of resulting differences on secondary structure determination under varying sample conditions (temperature, pH) are demonstrated and discussed for globular proteins and especially IDPs.

Pure shift amide detection in conventional and TROSY-type experiments of 13C,15N-labeled proteins.

Large coupling networks in uniformly 13C,15N-labeled biomolecules induce broad multiplets that even in flexible proteins are frequently not recognized as such. The reason is that given multiplets typically consist of a large number of individual resonances that result in a single broad line, in which individual components are no longer resolved. We here introduce a real-time pure shift acquisition scheme for the detection of amide protons which is based on 13C-BIRDr,X. As a result the full homo- and heteronuclear coupling network can be suppressed at low power leading to real singlets at substantially improved resolution and uncompromised sensitivity. The method is tested on a small globular and an intrinsically disordered protein (IDP) where the average spectral resolution is increased by a factor of ~ 2 and higher. Equally important, the approach works without saturation of water magnetization for solvent suppression and exchanging amide protons are not affected by saturation transfer.

The Disordered EZH2 Loop: Atomic Level Characterization by 1HN- and 1Hα-Detected NMR Approaches, Interaction with the Long Noncoding HOTAIR RNA.

The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the 1HN-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, 1Hα-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407-Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR140 (1-140 nt) highlight that the Thr367-Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein-RNA interaction as a "fuzzy" complex.

Monitoring Protein Global and Local Parameters in Unfolding and Binding Studies: The Extended Applicability of the Diffusion Coefficient─Molecular Size Empirical Relations.

Protein unfolding and denaturation are main issues in biochemical and pharmaceutical research. Using a global parameter, the translational diffusion coefficient D, folded, unfolded, and intrinsically disordered proteins of a given molar mass M can be distinguished based on their distinct hydrodynamic properties. For broader applications, we provide generalized, PFG-NMR-based empirical D-M relations validated at different temperatures and ready to use with the corresponding corrections in different media. We demonstrate that these relations enable a more accurate molecular mass determination and show fewer potential errors than those of the common methods based on small-molecular diffusion standards. We monitor unfolding of three model proteins using 8 M urea and dimethyl sulfoxide (DMSO)-water mixtures as denaturing agents, highlighting the effect of disulfide bonds. Denaturation in 8 M urea is pH-dependent; in addition, for proteins with highly stable disulfide bonds, a reducing agent (TCEP) is required to achieve complete unfolding. Regarding the effect of local parameters, we show that at low DMSO concentrations─common conditions in pharmaceutical binding studies─the PFG-NMR-derived global parameters are not significantly affected. Still, the atomic environments can change, and the bound solvent molecule can inhibit the binding of a partner molecule. Using proteins with natural isotopic abundance, this effect can be proven by fast 1H-15N 2D correlation spectra. Our results enable fast and easy estimation of protein molecular mass and the degree of folding in various media; moreover, the effect of the cosolvent on the atomic-level structure can be traced without the need of isotope labeling.

Selective 1 Hα NMR Methods Reveal Functionally Relevant Proline cis/trans Isomers in Intrinsically Disordered Proteins: Characterization of Minor Forms, Effects of Phosphorylation, and Occurrence in Proteome.

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two 1 Hα -detected, proline selective, real-time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4-15 % amounts; moreover, we show the consequences of CK2 phosphorylation on the cis/trans-proline equilibrium. Using our results and available literature data we perform a statistical analysis on how the amino acid type effects the cis/trans-proline distribution. The methods are applicable under physiological conditions, they can contribute to find key proline isomers in proteins, and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes.

Novel Lysine-Rich Delivery Peptides of Plant Origin ERD and Human S100: The Effect of Carboxyfluorescein Conjugation, Influence of Aromatic and Proline Residues, Cellular Internalization, and Penetration Ability.

The need for novel drug delivery peptides is an important issue of the modern pharmaceutical research. Here, we test K-rich peptides from plant dehydrin ERD14 (ERD-A, ERD-B, and ERD-C) and the C-terminal CPP-resembling region of S100A4 (S100) using the 5(6)-carboxyfluorescein (Cf) tag at the N-terminus. Via a combined pH-dependent NMR and fluorescence study, we analyze the effect of the Cf conjugation/modification on the structural behavior, separately investigating the (5)-Cf and (6)-Cf forms. Flow cytometry results show that all peptides internalize; however, there is a slight difference between the cellular internalization of (5)- and (6)-Cf-peptides. We indicate the possible importance of residues with an aromatic sidechain and proline. We prove that ERD-A localizes mostly in the cytosol, ERD-B and S100 have partial colocalization with lysosomal staining, and ERD-C mainly localizes within vesicle-like compartments, while the uptake mechanism mainly occurs through energy-dependent paths.

Power of Pure Shift HαCα Correlations: A Way to Characterize Biomolecules under Physiological Conditions.

Intrinsically disordered proteins (IDPs) constitute an important class of biomolecules with high flexibility. Atomic-resolution studies for these molecules are essentially limited to NMR spectroscopy, which should be performed under physiological pH and temperature to populate relevant conformational ensembles. In this context, however, fundamental problems arise with established triple resonance NMR experiments: high solvent accessibility of IDPs promotes water exchange, which disfavors classical amide 1H-detection, while 13C-detection suffers from significantly reduced sensitivity. A favorable alternative, the conventional detection of nonexchangeable 1Hα, so far resulted in broad signals with insufficient resolution and sensitivity. To overcome this, we introduce here a selective Hα,Cα-correlating pure shift detection scheme, the selective Hα,Cα-HSQC (SHACA-HSQC), using extensive hetero- and homonuclear decoupling applicable to aqueous samples (≥90% H2O) and tested on small molecules and proteins. SHACA-HSQC spectra acquired on IDPs provide uncompromised resolution and sensitivity (up to fivefold increased S/N compared to the standard 1H,13C-HSQC), as shown for resonance distinction and unambiguous assignment on the disordered transactivation domain of the tumor suppressor p53, α-synuclein, and folded ubiquitin. The detection scheme can be implemented in any 1Hα-detected triple resonance experiment and may also form the basis for the detection of isotope-labeled markers in biological studies or compound libraries.

Tumor-Suppressor p53TAD1-60 Forms a Fuzzy Complex with Metastasis-Associated S100A4: Structural Insights and Dynamics by an NMR/MD Approach.

Conformationally flexible protein complexes represent a major challenge for structural and dynamical studies. We present herein a method based on a hybrid NMR/MD approach to characterize the complex formed between the disordered p53TAD1-60 and the metastasis-associated S100A4. Disorder-to-order transitions of both TAD1 and TAD2 subdomains upon interaction is detected. Still, p53TAD1-60 remains highly flexible in the bound form, with residues L26, M40, and W53 being anchored to identical hydrophobic pockets of the S100A4 monomer chains. In the resulting "fuzzy" complex, the clamp-like binding of p53TAD1-60 relies on specific hydrophobic anchors and on the existence of extended flexible segments. Our results demonstrate that structural and dynamical NMR parameters (cumulative Δδ, SSP, temperature coefficients, relaxation time, hetNOE) combined with MD simulations can be used to build a structural model even if, due to high flexibility, the classical solution structure calculation is not possible.

Anionic food color tartrazine enhances antibacterial efficacy of histatin-derived peptide DHVAR4 by fine-tuning its membrane activity.

Here it is demonstrated how some anionic food additives commonly used in our diet, such as tartrazine (TZ), bind to DHVAR4, an antimicrobial peptide (AMP) derived from oral host defense peptides, resulting in significantly fostered toxic activity against both Gram-positive and Gram-negative bacteria, but not against mammalian cells. Biophysical studies on the DHVAR4-TZ interaction indicate that initially large, positively charged aggregates are formed, but in the presence of lipid bilayers, they rather associate with the membrane surface. In contrast to synergistic effects observed for mixed antibacterial compounds, this is a principally different mechanism, where TZ directly acts on the membrane-associated AMP promoting its biologically active helical conformation. Model vesicle studies show that compared to dye-free DHVAR4, peptide-TZ complexes are more prone to form H-bonds with the phosphate ester moiety of the bilayer head-group region resulting in more controlled bilayer fusion mechanism and concerted severe cell damage. AMPs are considered as promising compounds to combat formidable antibiotic-resistant bacterial infections; however, we know very little on their in vivo actions, especially on how they interact with other chemical agents. The current example illustrates how food dyes can modulate AMP activity, which is hoped to inspire improved therapies against microbial infections in the alimentary tract. Results also imply that the structure and function of natural AMPs could be manipulated by small compounds, which may also offer a new strategic concept for the future design of peptide-based antimicrobials.

Real-time pure shift measurements for uniformly isotope-labeled molecules using X-selective BIRD homonuclear decoupling.

We introduce a novel selective inversion element for chunked homonuclear decoupling that combines isotope selection via BIRD-filtering with band-selective inversion on the X-heteronucleus and allows efficient real-time decoupling of homonuclear and heteronuclear couplings. It is especially suitable for uniformly isotope-labeled compounds. We discuss in detail the inversion element based on band-selective refocusing on the X-nuclei (BASEREX), highlighting in particular the role of appropriate band-selective shaped refocusing pulses and the application of broadband X-pulses for an effective BIRDd element during homodecoupling. The approach is experimentally verified and studied in detail using uniformly 13C-labeled glucose and a uniformly 15N,13C-labeled amino acid mixture.

Quantitative, Diffusion NMR Based Analytical Tool To Distinguish Folded, Disordered, and Denatured Biomolecules.

The key questions in folding studies are the protein dimensions and the degree of folding. These properties are best characterized by the self-diffusion coefficients D determining the hydrodynamic dimensions. In our present study, we derive empirical variations of D as a function of molecular mass M that distinguish folded, intrinsically disordered, and urea-denatured biomolecules. Reliable D values are obtained from diffusion NMR measurements performed under identical conditions using a representative set of proteins/peptides with diverse amino acid sequence and length. The established relations are easy to use analytical tools for molecular mass analysis and aggregation studies as well. Deriving equations under denaturing conditions has several pitfalls, and here, we provide a simple quantitative method for estimating the debated end point of denaturation, while already the 1D 1H spectrum gives a qualitative picture of the collapsed, denatured structure. Data indicate that the intrinsically disordered proteins have a similar behavior as synthetic polymers and urea-denatured proteins.

Directed Evolution of Canonical Loops and Their Swapping between Unrelated Serine Proteinase Inhibitors Disprove the Interscaffolding Additivity Model.

Reversible serine proteinase inhibitors comprise 18 unrelated families. Each family has a distinct representative structure but contains a surface loop that adopts the same, canonical conformation in the enzyme-inhibitor complex. The Laskowski mechanism universally applies for the action of all canonical inhibitors independent of their scaffold, but it has two nontrivial extrapolations. Intrascaffolding additivity states that all enzyme-contacting loop residues act independently of each other, while interscaffolding additivity claims that these residues act independently of the scaffold. These theories have great importance for engineering proteinase inhibitors but have not been comprehensively challenged. Therefore, we tested the interscaffolding additivity theory by hard-randomizing all enzyme-contacting canonical loop positions of a Kazal- and a Pacifastin-scaffold inhibitor, displaying the variants on M13 phage, and selecting the libraries on trypsin and chymotrypsin. Directed evolution delivered different patterns on both scaffolds against both enzymes, which contradicts interscaffolding additivity. To quantitatively assess the extent of non-additivity, we measured the affinities of the optimal binding loop variants and their binding loop-swapped versions. While optimal variants have picomolar affinities, swapping the evolved loops results in up to 200,000-fold affinity loss. To decipher the underlying causes, we characterized the stability, overall structure and dynamics of the inhibitors with differential scanning calorimetry, circular dichroism and NMR spectroscopy and molecular dynamic simulations. These studies revealed that the foreign loop destabilizes the lower-stability Pacifastin scaffold, while the higher-stability Kazal scaffold distorts the foreign loop. Our findings disprove interscaffolding additivity and show that loop and scaffold form one integrated unit that needs to be coevolved to provide high-affinity inhibition.

Multiple S100 protein isoforms and C-terminal phosphorylation contribute to the paralog-selective regulation of nonmuscle myosin 2 filaments.

Nonmuscle myosin 2 (NM2) has three paralogs in mammals, NM2A, NM2B, and NM2C, which have both unique and overlapping functions in cell migration, formation of cell-cell adhesions, and cell polarity. Their assembly into homo- and heterotypic bipolar filaments in living cells is primarily regulated by phosphorylation of the N-terminally bound regulatory light chain. Here, we present evidence that the equilibrium between these filaments and single NM2A and NM2B molecules can be controlled via S100 calcium-binding protein interactions and phosphorylation at the C-terminal end of the heavy chains. Furthermore, we show that in addition to S100A4, other members of the S100 family can also mediate disassembly of homotypic NM2A filaments. Importantly, these proteins can selectively remove NM2A molecules from heterotypic filaments. We also found that tail phosphorylation (at Ser-1956 and Ser-1975) of NM2B by casein kinase 2, as well as phosphomimetic substitutions at sites targeted by protein kinase C (PKC) and transient receptor potential cation channel subfamily M member 7 (TRPM7), down-regulates filament assembly in an additive fashion. Tail phosphorylation of NM2A had a comparatively minor effect on filament stability. S100 binding and tail phosphorylation therefore preferentially disassemble NM2A and NM2B, respectively. These two distinct mechanisms are likely to contribute to the temporal and spatial sorting of the two NM2 paralogs within heterotypic filaments. The existence of multiple NM2A-depolymerizing S100 paralogs offers the potential for diverse regulatory inputs modulating NM2A filament disassembly in cells and provides functional redundancy under both physiological and pathological conditions.

New Antimicrobial Potential and Structural Properties of PAFB: A Cationic, Cysteine-Rich Protein from Penicillium chrysogenum Q176.

Small, cysteine-rich and cationic proteins with antimicrobial activity are produced by diverse organisms of all kingdoms and represent promising molecules for drug development. The ancestor of all industrial penicillin producing strains, the ascomycete Penicillium chryosgenum Q176, secretes the extensively studied antifungal protein PAF. However, the genome of this strain harbours at least two more genes that code for other small, cysteine-rich and cationic proteins with potential antifungal activity. In this study, we characterized the pafB gene product that shows high similarity to PgAFP from P. chrysogenum R42C. Although abundant and timely regulated pafB gene transcripts were detected, we could not identify PAFB in the culture broth of P. chrysogenum Q176. Therefore, we applied a P. chrysogenum-based expression system to produce sufficient amounts of recombinant PAFB to address unanswered questions concerning the structure and antimicrobial function. Nuclear magnetic resonance (NMR)-based analyses revealed a compact ß-folded structure, comprising five ß-strands connected by four solvent exposed and flexible loops and an "abcabc" disulphide bond pattern. We identified PAFB as an inhibitor of growth of human pathogenic moulds and yeasts. Furthermore, we document for the first time an anti-viral activity for two members of the small, cysteine-rich and cationic protein group from ascomycetes.

Dynamic control of RSK complexes by phosphoswitch-based regulation.

Assembly and disassembly of protein-protein complexes needs to be dynamically controlled and phosphoswitches based on linear motifs are crucial in this process. Extracellular signal-regulated kinase 2 (ERK2) recognizes a linear-binding motif at the C-terminal tail (CTT) of ribosomal S6 kinase 1 (RSK1), leading to phosphorylation and subsequent activation of RSK1. The CTT also contains a classical PDZ domain-binding motif which binds RSK substrates (e.g. MAGI-1). We show that autophosphorylation of the disordered CTT promotes the formation of an intramolecular charge clamp, which efficiently masks critical residues and indirectly hinders ERK binding. Thus, RSK1 CTT operates as an autoregulated phosphoswitch: its phosphorylation at specific sites affects its protein-binding capacity and its conformational dynamics. These biochemical feedbacks, which form the structural basis for the rapid dissociation of ERK2-RSK1 and RSK1-PDZ substrate complexes under sustained epidermal growth factor (EGF) stimulation, were structurally characterized and validated in living cells. Overall, conformational changes induced by phosphorylation in disordered regions of protein kinases, coupled to allosteric events occurring in the kinase domain cores, may provide mechanisms that contribute to the emergence of complex signaling activities. In addition, we show that phosphoswitches based on linear motifs can be functionally classified as ON and OFF protein-protein interaction switches or dimmers, depending on the specific positioning of phosphorylation target sites in relation to functional linear-binding motifs. Moreover, interaction of phosphorylated residues with positively charged residues in disordered regions is likely to be a common mechanism of phosphoregulation. DATABASE: Structural data are available in the PDB database under the accession numbers 5N7D, 5N7F and 5N7G. NMR spectral assignation data are available in the BMRB database under the accession numbers 27213 and 27214.

Ezrin interacts with S100A4 via both its N- and C-terminal domains.

Ezrin belongs to the ERM (ezrin, radixin, moesin) protein family that has a role in cell morphology changes, adhesion and migration as an organizer of the cortical cytoskeleton by linking actin filaments to the apical membrane of epithelial cells. It is highly expressed in a variety of human cancers and promotes metastasis. Members of the Ca2+-binding EF-hand containing S100 proteins have similar pathological properties; they are overexpressed in cancer cells and involved in metastatic processes. In this study, using tryptophan fluorescence and stopped-flow kinetics, we show that S100A4 binds to the N-terminal ERM domain (N-ERMAD) of ezrin with a micromolar affinity. The binding involves the F2 lobe of the N-ERMAD and follows an induced fit kinetic mechanism. Interestingly, S100A4 binds also to the unstructured C-terminal actin binding domain (C-ERMAD) with similar affinity. Using NMR spectroscopy, we characterized the complex of S100A4 with the C-ERMAD and demonstrate that no ternary complex is simultaneously formed with the two ezrin domains. Furthermore, we show that S100A4 co-localizes with ezrin in HEK-293T cells. However, S100A4 very weakly binds to full-length ezrin in vitro indicating that the interaction of S100A4 with ezrin requires other regulatory events such as protein phosphorylation and/or membrane binding, shifting the conformational equilibrium of ezrin towards the open state. As both proteins play an important role in promoting metastasis, the characterization of their interaction could shed more light on the molecular events contributing to this pathological process.