Including the Ensemble of Unstructured Conformations in the Analysis of Protein's Native State by High-Pressure NMR Spectroscopy.

The analysis of pressure induced changes in the chemical shift of proteins allows statements on structural fluctuations proteins exhibit at ambient pressure. The inherent issue of separating general pressure effects from structural related effects on the pressure dependence of chemical shifts has so far been addressed by considering the characteristics of random coil peptides on increasing pressure. In this work, chemically and pressure denatured states of the cold shock protein B from Bacillus subtilis (BsCspB) have been assigned in 2D 1H-15N HSQC NMR spectra and their dependence on increasing hydrostatic pressure has been evaluated. The pressure denatured polypeptide chain has been used to separate general from structural related effects on 1H and 15N chemical shifts of native BsCspB and the implications on the interpretation of pressure induced changes in the chemical shift regarding the structure of BsCspB are discussed. It has been found that the ensemble of unstructured conformations of BsCspB shows different responses to increasing pressure than random coil peptides do. Thus, the approach used for considering the general effects that arise when hydrostatic pressure increases changes the structural conclusions that are drawn from high pressure NMR spectroscopic experiments that rely on the analysis of chemical shifts.

Targeted Preparation and NMR Spectroscopic Characterization of Lys11-Linked Ubiquitin Trimers.

Ubiquitylation refers to the attachment of mono- or poly-ubiquitin molecules to a substrate protein. To shield ubiquitin chains against potential hydrolysis, a facile, click-chemistry based approach was recently established for the generation of site-specifically conjugated ubiquitin dimers relying on triazole-linkage. Here, the preparation of such ubiquitin chains was advanced by the generation of homotypic Lys11-linked ubiquitin trimers considering an isotopic labeling scheme in a moiety-wise manner. The structural and dynamical impact on the ubiquitin unit at proximal, central, or distal position that is potentially invoked by the respective other two moieties was systematically probed by heteronuclear high-resolution NMR spectroscopic approaches. As a result, conjugating a third ubiquitin moiety to the proximal or distal site of a ubiquitin dimer does not alter structural and dynamical characteristics as it has been seen for ubiquitin dimers. This observation suggests that recognition of a homotypically assembled ubiquitin chain by a potential substrate is primarily done by screening the length of a ubiquitin chain rather than relying on subtle changes in structure or dynamic properties of single ubiquitin moieties composing the chain.

Specifying conformational heterogeneity of multi-domain proteins at atomic resolution.

The conformational landscape of multi-domain proteins is inherently linked to their specific functions. This also holds for polyubiquitin chains that are assembled by two or more ubiquitin domains connected by a flexible linker thus showing a large interdomain mobility. However, molecular recognition and signal transduction are associated with particular conformational substates that are populated in solution. Here, we apply high-resolution NMR spectroscopy in combination with dual-scale MD simulations to explore the conformational space of K6-, K29-, and K33-linked diubiquitin molecules. The conformational ensembles are evaluated utilizing a paramagnetic cosolute reporting on solvent exposure plus a set of complementary NMR parameters. This approach unravels a conformational heterogeneity of diubiquitins and explains the diversity of structural models that have been determined for K6-, K29-, and K33-linked diubiquitins in free and ligand-bound states so far. We propose a general application of the approach developed here to demystify multi-domain proteins occurring in nature.

Distinct growth regimes of α-synuclein amyloid elongation.

Addition of amyloid seeds to aggregation-prone monomers allows for amyloid fiber growth (elongation) omitting slow nucleation. We here combine Thioflavin T fluorescence (probing formation of amyloids) and solution-state NMR spectroscopy (probing disappearance of monomers) to assess elongation kinetics of the amyloidogenic protein, α-synuclein, for which aggregation is linked to Parkinson's disease. We found that both spectroscopic detection methods give similar kinetic results, which can be fitted by applying double exponential decay functions. When the origin of the two-phase behavior was analyzed by mathematical modeling, parallel paths as well as stop-and-go behavior were excluded as possible explanations. Instead, supported by previous theory, the experimental elongation data reveal distinct kinetic regimes that depend on instantaneous monomer concentration. At low monomer concentrations (toward end of experiments), amyloid growth is limited by conformational changes resulting in ß-strand alignments. At the higher monomer concentrations (initial time points of experiments), growth occurs rapidly by incorporating monomers that have not successfully completed the conformational search. The presence of a fast disordered elongation regime at high monomer concentrations agrees with coarse-grained simulations and theory but has not been detected experimentally before. Our results may be related to the wide range of amyloid folds observed.

The Mycobacterium tuberculosis protein tyrosine phosphatase MptpA features a pH dependent activity overlapping the bacterium sensitivity to acidic conditions.

The Mycobacterium tuberculosis low-molecular weight protein tyrosine phosphatase (MptpA) is responsible for the inhibition of phagosome-lysosome fusion and is essential for the bacterium pathogenicity. This inhibition implies that M. tuberculosis is not exposed to a strongly acidic environment in vivo, enabling successful propagation in host cells. Remarkably, MptpA has been previously structurally and functionally investigated, with special emphasis devoted to the enzyme properties at pH 8.0. Considering that the virulence of M. tuberculosis is strictly dependent on the avoidance of acidic conditions in vivo, we analysed the pH-dependence of the structural and catalytic properties of MptpA. Here we show that this enzyme undergoes pronounced conformational rearrangements when exposed to acidic pH conditions, inducing a severe decrease of the enzymatic catalytic efficiency at the expense of phosphotyrosine (pTyr). In particular, a mild decrease of pH from 6.5 to 6.0 triggers a significant increase of K0.5 of MptpA for phosphotyrosine, the phosphate group of which we determined to feature a pKa2 equal to 5.7. Surface plasmon resonance experiments confirmed that MptpA binds poorly to pTyr at pH values < 6.5. Notably, the effectiveness of the MptpA competitive inhibitor L335-M34 at pH 6 does largely outperform the inhibition exerted at neutral or alkaline pH values. Overall, our observations indicate a pronounced sensitivity of MptpA to acidic pH conditions, and suggest the search for competitive inhibitors bearing a negatively charged group featuring pKa values lower than that of the substrate phosphate group.

Lipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking.

The abundance of plasma membrane-resident receptors and transporters has to be tightly regulated by ubiquitin-mediated endosomal degradation for the proper coordination of environmental stimuli and intracellular signaling. Arabidopsis OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 are plasma membrane-localized deubiquitylating enzymes (DUBs) that bind to phospholipids through a polybasic motif in the OTU domain. Here we show that the DUB activity of OTU11 and OTU12 towards K63-linked ubiquitin is stimulated by binding to lipid membranes containing anionic lipids. In addition, we show that the DUB activity of OTU11 against K6- and K11-linkages is also stimulated by anionic lipids, and that OTU11 and OTU12 can modulate the endosomal degradation of a model cargo and the auxin efflux transporter PIN2-GFP in vivo. Our results suggest that the catalytic activity of OTU11 and OTU12 is tightly connected to their ability to bind membranes and that OTU11 and OTU12 are involved in the fine-tuning of plasma membrane proteins in Arabidopsis.

Electrostatic and steric effects underlie acetylation-induced changes in ubiquitin structure and function.

Covalent attachment of ubiquitin (Ub) to proteins is a highly versatile posttranslational modification. Moreover, Ub is not only a modifier but itself is modified by phosphorylation and lysine acetylation. However, the functional consequences of Ub acetylation are poorly understood. By generation and comprehensive characterization of all seven possible mono-acetylated Ub variants, we show that each acetylation site has a particular impact on Ub structure. This is reflected in selective usage of the acetylated variants by different E3 ligases and overlapping but distinct interactomes, linking different acetylated variants to different cellular pathways. Notably, not only electrostatic but also steric effects contribute to acetylation-induced changes in Ub structure and, thus, function. Finally, we provide evidence that p300 acts as a position-specific Ub acetyltransferase and HDAC6 as a general Ub deacetylase. Our findings provide intimate insights into the structural and functional consequences of Ub acetylation and highlight the general importance of Ub acetylation.

Identification of novel functional mini-receptors by combinatorial screening of split-WW domains.

ß-Sheet motifs such as the WW domain are increasingly being explored as building blocks for synthetic biological applications. Since the sequence-structure relationships of ß-sheet motifs are generally complex compared to the well-studied α-helical coiled coil (CC), other approaches such as combinatorial screening should be included to vary the function of the peptide. In this study, we present a combinatorial approach to identify novel functional mini-proteins based on the WW-domain scaffold, which takes advantage of the successful reconstitution of the fragmented WW domain of hPin1 (hPin1WW) by CC association. Fragmentation of hPin1WW was performed in both loop 1 (CC-hPin1WW-L1) and loop 2 (CC-hPin1WW-L2), and the respective fragments were linked to the strands of an antiparallel heterodimeric CC. Structural analysis by CD and NMR spectroscopy revealed structural reconstitution of the WW-domain scaffold only in CC-hPin1WW-L1, but not in CC-hPin1WW-L2. Furthermore, by using 1H-15N HSQC NMR, fluorescence and CD spectroscopy, we demonstrated that binding properties of fragmented hPin1WW in CC-hPin1WW-L1 were fully restored by CC association. To demonstrate the power of this approach as a combinatorial screening platform, we synthesized a four-by-six library of N- and C-terminal hPin1WW-CC peptide fragments that was screened for a WW domain that preferentially binds to ATP over cAMP, phophocholine, or IP6. Using this screening platform, we identified one WW domain, which specifically binds ATP, and a phosphorylcholine-specific WW-based mini-receptor, both having binding dissociation constants in the lower micromolar range.

Completing the family of human Eps15 homology domains: Solution structure of the internal Eps15 homology domain of γ-synergin.

Eps15 homology (EH) domains are universal interaction domains to establish networks of protein-protein interactions in the cell. These networks mainly coordinate cellular functions including endocytosis, actin remodeling, and other intracellular signaling pathways. They are well characterized in structural terms, except for the internal EH domain from human γ-synergin (EHγ). Here, we complete the family of EH domain structures by determining the solution structure of the EHγ domain. The structural ensemble follows the canonical EH domain fold and the identified binding site is similar to other known EH domains. But EHγ differs significantly in the N- and C-terminal regions. The N-terminal α-helix is shortened compared to known homologues, while the C-terminal one is fully formed. A significant proportion of the remaining N- and C-terminal regions are well structured, a feature not seen in other EH domains. Single mutations in both the N-terminal and the C-terminal structured extensions lead to the loss of the distinct three-dimensional fold and turn EHγ into a molten globule like state. Therefore, we propose that the structural extensions in EHγ function as a clamp and are undoubtedly required to maintain its tertiary fold.

Switchable Zinc(II)-Responsive Globular ß-Sheet Peptide.

The natural function of many proteins depends on their ability to switch their conformation driven by environmental changes. In this work, we present a small, monomeric ß-sheet peptide that switches between a molten globule and a folded state through Zn(II) binding. The solvent-exposed hydrophobic core on the ß-sheet surface was substituted by a His3-site, whereas the internal hydrophobic core was left intact. Zn(II) is specifically recognized by the peptide relative to other divalent metal ions, binds in the lower micromolar range, and can be removed and re-added without denaturation of the peptide. In addition, the peptide is fully pH-switchable, has a pKa of about 6, and survives several cycles of acidification and neutralization. In-depth structural characterization of the switch was achieved by concerted application of circular dichroism (CD) and multinuclear NMR spectroscopy. Thus, this study represents a viable approach toward a globular ß-sheet Zn(II) mini-receptor prototype.

Structural and functional consequences of NEDD8 phosphorylation.

Ubiquitin (Ub) and Ub-like proteins (Ubls) such as NEDD8 are best known for their function as covalent modifiers of other proteins but they are also themselves subject to post-translational modifications including phosphorylation. While functions of phosphorylated Ub (pUb) have been characterized, the consequences of Ubl phosphorylation remain unclear. Here we report that NEDD8 can be phosphorylated at S65 - the same site as Ub - and that S65 phosphorylation affects the structural dynamics of NEDD8 and Ub in a similar manner. While both pUb and phosphorylated NEDD8 (pNEDD8) can allosterically activate the Ub ligase Parkin, they have different protein interactomes that in turn are distinct from those of unmodified Ub and NEDD8. Among the preferential pNEDD8 interactors are HSP70 family members and we show that pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings highlight the general importance of Ub/NEDD8 phosphorylation and support the notion that the function of pUb/pNEDD8 does not require their covalent attachment to other proteins.

Impact of crowded environments on binding between protein and single-stranded DNA.

The concept of Molecular Crowding depicts the high density of diverse molecules present in the cellular interior. Here, we determine the impact of low molecular weight and larger molecules on binding capacity of single-stranded DNA (ssDNA) to the cold shock protein B (CspB). Whereas structural features of ssDNA-bound CspB are fully conserved in crowded environments as probed by high-resolution NMR spectroscopy, intrinsic fluorescence quenching experiments reveal subtle changes in equilibrium affinity. Kinetic stopped-flow data showed that DNA-to-protein association is significantly retarded independent of choice of the molecule that is added to the solution, but dissociation depends in a nontrivial way on its size and chemical characteristics. Thus, for this DNA-protein interaction, excluded volume effect does not play the dominant role but instead observed effects are dictated by the chemical properties of the crowder. We propose that surrounding molecules are capable of specific modification of the protein's hydration shell via soft interactions that, in turn, tune protein-ligand binding dynamics and affinity.

Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate.

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution 19 F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.

Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism.

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.

Template-assisted design of monomeric polyQ models to unravel the unique role of glutamine side chains in disease-related aggregation.

Expanded polyglutamine (polyQ) sequences cause numerous neurodegenerative diseases which are accompanied by the formation of polyQ fibrils. The unique role of glutamines in the aggregation onset is undoubtedly accepted and a lot structural data of the fibrils have been acquired, however side-chain specific structural dynamics inducing oligomerization are not well understood yet. To analyze spectroscopically the nucleation process, we designed various template-assisted glutamine-rich ß-hairpin monomers mimicking the structural motif of a polyQ fibril. In a top-down strategy, we use a template which forms a well-defined stable hairpin in solution, insert polyQ-rich sequences into each strand and monitor the effects of individual glutamines by NMR, CD and IR spectroscopic approaches. The design was further advanced by alternating glutamines with other amino acids (T, W, E, K), thereby enhancing the solubility and increasing the number of cross-strand interacting glutamine side chains. Our spectroscopic studies reveal a decreasing hairpin stability with increased glutamine content and demonstrate the enormous impact of only a few glutamines - far below the disease threshold - to destabilize structure. Furthermore, we could access sub-ms conformational dynamics of monomeric polyQ-rich peptides by laser-excited temperature-jump IR spectroscopy. Both, the increased number of interacting glutamines and higher concentrations are key parameters to induce oligomerization. Concentration-dependent time-resolved IR measurements indicate an additional slower kinetic phase upon oligomer formation. The here presented peptide models enable spectroscopic molecular analyses to distinguish between monomer and oligomer dynamics in the early steps of polyQ fibril formation and in a side-chain specific manner.

All atom insights into the impact of crowded environments on protein stability by NMR spectroscopy.

The high density of macromolecules affecting proteins due to volume exclusion has been discussed in theory but numerous in vivo experiments cannot be sufficiently understood taking only pure entropic stabilization into account. Here, we show that the thermodynamic stability of a beta barrel protein increases equally at all atomic levels comparing crowded environments with dilute conditions by applying multidimensional high-resolution NMR spectroscopy in a systematic manner. Different crowding agents evoke a pure stabilization cooperatively and do not disturb the surface or integrity of the protein fold. The here developed methodology provides a solid base that can be easily expanded to incorporate e.g. binding partners to recognize functional consequences of crowded conditions. Our results are relevant to research projects targeting soluble proteins in vivo as it can be anticipated that their thermodynamic stability increase comparably and has consequently to be taken into account to coherently understand intracellular processes.

Synthesis of nickel hexacyanoferrate nanocubes with tuneable dimensions via temperature-controlled Ni2+-citrate complexation.

The citrate-assisted growth of nickel hexacyanoferrate (NiHCF) nanocubes was investigated. Control over the complexation of Ni2+ ions with citrate at different temperatures enabled fine tuning of the nanocrystal (NC) dimensions and their self-assembly into mesocrystals. Our results introduce new concepts towards the synthesis of NiHCF NCs, potentially applicable to other members of the Prussian blue analogues family.

Insights into Protein Stability in Cell Lysate by 19 F NMR Spectroscopy.

In living organisms, protein folding and function take place in an inhomogeneous, highly crowded environment possessing a concentration of diverse macromolecules of up to 400 g/L. It has been shown that the intracellular environment has a pronounced effect on the stability, dynamics and function of the protein under study, and has for this reason to be considered. However, most protein studies neglect the presence of these macromolecules. Consequently, we probe here the overall thermodynamic stability of cold shock protein B from Bacillus subtilis (BsCspB) in cell lysate. We found that an increase in cell lysate concentration causes a monotonic increase in the thermodynamic stability of BsCspB. This result strongly underlines the importance of considering the biological environment when inherent protein parameters are quantitatively determined. Moreover, we demonstrate that targeted application of 19 F NMR spectroscopy operates as an ideal tool for protein studies performed in complex cellular surroundings.

What does fluorine do to a protein? Thermodynamic, and highly-resolved structural insights into fluorine-labelled variants of the cold shock protein.

Fluorine labelling represents one promising approach to study proteins in their native environment due to efficient suppressing of background signals. Here, we systematically probe inherent thermodynamic and structural characteristics of the Cold shock protein B from Bacillus subtilis (BsCspB) upon fluorine labelling. A sophisticated combination of fluorescence and NMR experiments has been applied to elucidate potential perturbations due to insertion of fluorine into the protein. We show that single fluorine labelling of phenylalanine or tryptophan residues has neither significant impact on thermodynamic stability nor on folding kinetics compared to wild type BsCspB. Structure determination of fluorinated phenylalanine and tryptophan labelled BsCspB using X-ray crystallography reveals no displacements even for the orientation of fluorinated aromatic side chains in comparison to wild type BsCspB. Hence we propose that single fluorinated phenylalanine and tryptophan residues used for protein labelling may serve as ideal probes to reliably characterize inherent features of proteins that are present in a highly biological context like the cell.

Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers.

Ubiquitylation is an eminent posttranslational modification referring to the covalent attachment of single ubiquitin molecules or polyubiquitin chains to a target protein dictating the fate of such labeled polypeptide chains. Here, we have biochemically produced artificially Lys11-, and Lys27-, and Lys63-linked ubiquitin dimers based on click-chemistry generating milligram quantities in high purity. We show that the artificial linkage used for the conjugation of two ubiquitin moieties represents a fully reliable surrogate of the natural isopeptide bond by acquiring highly resolved nuclear magnetic resonance (NMR) spectroscopic data including ligand binding studies. Extensive coarse grained and atomistic molecular dynamics (MD) simulations allow to extract structures representing the ensemble of domain-domain conformations used to verify the experimental data. Advantageously, this methodology does not require individual isotopic labeling of both ubiquitin moieties as NMR data have been acquired on the isotopically labeled proximal moiety and complementary MD simulations have been used to fully interpret the experimental data in terms of domain-domain conformation. This combined approach intertwining NMR spectroscopy with MD simulations makes it possible to describe the conformational space non-canonically Lys11-, and Lys27-linked ubiquitin dimers occupy in a solution averaged ensemble by taking atomically resolved information representing all residues in ubiquitin dimers into account.