Multivalent binding kinetics resolved by fluorescence proximity sensing.

Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.

pH- and concentration-dependent supramolecular assembly of a fungal defensin plectasin variant into helical non-amyloid fibrils.

Self-assembly and fibril formation play important roles in protein behaviour. Amyloid fibril formation is well-studied due to its role in neurodegenerative diseases and characterized by refolding of the protein into predominantly ß-sheet form. However, much less is known about the assembly of proteins into other types of supramolecular structures. Using cryo-electron microscopy at a resolution of 1.97 Å, we show that a triple-mutant of the anti-microbial peptide plectasin, PPI42, assembles into helical non-amyloid fibrils. The in vitro anti-microbial activity was determined and shown to be enhanced compared to the wildtype. Plectasin contains a cysteine-stabilised α-helix-ß-sheet structure, which remains intact upon fibril formation. Two protofilaments form a right-handed protein fibril. The fibril formation is reversible and follows sigmoidal kinetics with a pH- and concentration dependent equilibrium between soluble monomer and protein fibril. This high-resolution structure reveals that α/ß proteins can natively assemble into fibrils.

Development of a fast screening method for selecting excipients in formulations using MD simulations, NMR and microscale thermophoresis.

Development of peptide therapeutics generally involves screening of excipients that inhibit peptide-peptide interactions, hence aggregation, and improve peptide stability. We used the therapeutic peptide plectasin to develop a fast screening method that combines microscale thermophoresis titration assays and molecular dynamics simulations to relatively rank the excipients with respect to binding affinity and to study key peptide-excipient interaction hotspots on a molecular level, respectively. Additionally, 1H-13C-HSQC NMR titration experiments were performed to validate the fast screening approach. The NMR results are in qualitative agreement with results from the fast screening method demonstrating that this approach can be reliably applied to other peptides and proteins as a fast screening method to relatively rank excipients and predict possible excipient binding sites.

The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide: Development of a Screening Protocol for Peptide Stability Screening.

Therapeutic peptides and proteins show enormous potential in the pharmaceutical market, but high costs in discovery and development are limiting factors so far. Single or multiple point mutations are commonly introduced in protein drugs to increase their binding affinity or selectivity. They can also induce adverse properties, which might be overlooked in a functional screen, such as a decreased colloidal or thermal stability, leading to problems in later stages of the development. In this study, we address the effect of point mutations on the stability of the 4.4 kDa antimicrobial peptide plectasin, as a case study. We combined a systematic high-throughput biophysical screen of the peptide thermal and colloidal stability using dynamic light scattering and differential scanning calorimetry with structure-based methods including small-angle X-ray scattering, analytical ultracentrifugation, and nuclear magnetic resonance spectroscopy. Additionally, we applied molecular dynamics simulations to link obtained protein stability parameters to the protein's molecular structure. Despite their predicted structural similarities, all four plectasin variants showed substantially different behavior in solution. We observed an increasing propensity of plectasin to aggregate at a higher pH, and the introduced mutations influenced the type of aggregation. Our strategy for systematically assessing the stability and aggregation of protein drugs is generally applicable and is of particular relevance, given the increasing number of protein drugs in development.

Albumin-neprilysin fusion protein: understanding stability using small angle X-ray scattering and molecular dynamic simulations.

Fusion technology is widely used in protein-drug development to increase activity, stability, and bioavailability of protein therapeutics. Fusion proteins, like any other type of biopharmaceuticals, need to remain stable during production and storage. Due to the high complexity and additional intramolecular interactions, it is not possible to predict the behavior of fusion proteins based on the behavior the individual proteins. Therefore, understanding the stability of fusion proteins on the molecular level is crucial for the development of biopharmaceuticals. The current study on the albumin-neprilysin (HSA-NEP) fusion protein uses a combination of thermal and chemical unfolding with small angle X-ray scattering and molecular dynamics simulations to show a correlation between decreasing stability and increasing repulsive interactions, which is unusual for most biopharmaceuticals. It is also seen that HSA-NEP is not fully flexible: it is present in both compact and extended conformations. Additionally, the volume fraction of each conformation changes with pH. Finally, the presence of NaCl and arginine increases stability at pH 6.5, but decreases stability at pH 5.0.

Conformational Stability Study of a Therapeutic Peptide Plectasin Using Molecular Dynamics Simulations in Combination with NMR.

Plectasin is a small, cysteine-rich peptide antibiotic which belongs to the class of antimicrobial peptides and has potential antibacterial activity against various Gram-positive bacteria. In the current study, the effect of pH and ionic strength (NaCl) on the conformational stability of plectasin variants has been investigated. At all physiochemical conditions, peptide secondary structures are intact throughout simulations. However, flexibility increases with pH because of the change in electrostatics around the distinct anionic tetrapeptide (9-12) stretch. Furthermore, plectasin interactions with NaCl were measured by determining the preferential interaction coefficients, Γ23. Generally, wild-type plectasin has higher preference for sodium ions as 9ASP is mutated in other variants. Overall, the Γ23 trend with pH for the two salt conditions remain the same for all variants predominately having accumulation of sodium ions around 10GLU and 12ASP. Insignificant changes in the overall peptide conformational stability are in agreement with the fact that plectasin has three cystines. Thermodynamic integration molecular dynamics simulations supplemented with nuclear magnetic resonance were employed to determine the degree of involvement of three different cystines to the overall structural integrity of the peptide. Both methods show the same order of cystine reduction and complete unfolding is observed only upon reduction of all cystines.

Interaction of RECQ4 and MCM10 is important for efficient DNA replication origin firing in human cells.

DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing.

Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose).

Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation.

Acetylation site specificities of lysine deacetylase inhibitors in human cells.

Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases.

Small angle X-ray scattering studies of mitochondrial glutaminase C reveal extended flexible regions, and link oligomeric state with enzyme activity.

Glutaminase C is a key metabolic enzyme, which is unregulated in many cancer systems and believed to play a central role in the Warburg effect, whereby cancer cells undergo changes to an altered metabolic profile. A long-standing hypothesis links enzymatic activity to the protein oligomeric state, hence the study of the solution behavior in general and the oligomer state in particular of glutaminase C is important for the understanding of the mechanism of protein activation and inhibition. In this report, this is extensively investigated in correlation to enzyme concentration or phosphate level, using a high-throughput microfluidic-mixing chip for the SAXS data collection, and we confirm that the oligomeric state correlates with activity. The in-depth solution behavior analysis further reveals the structural behavior of flexible regions of the protein in the dimeric, tetrameric and octameric state and investigates the C-terminal influence on the enzyme solution behavior. Our data enable SAXS-based rigid body modeling of the full-length tetramer states, thereby presenting the first ever experimentally derived structural model of mitochondrial glutaminase C including the N- and C-termini of the enzyme.

Functional proteomics defines the molecular switch underlying FGF receptor trafficking and cellular outputs.

The stimulation of fibroblast growth factor receptors (FGFRs) with distinct FGF ligands generates specific cellular responses. However, the mechanisms underlying this paradigm have remained elusive. Here, we show that FGF-7 stimulation leads to FGFR2b degradation and, ultimately, cell proliferation, whereas FGF-10 promotes receptor recycling and cell migration. By combining mass-spectrometry-based quantitative proteomics with fluorescence microscopy and biochemical methods, we find that FGF-10 specifically induces the rapid phosphorylation of tyrosine (Y) 734 on FGFR2b, which leads to PI3K and SH3BP4 recruitment. This complex is crucial for FGFR2b recycling and responses, given that FGF-10 stimulation of either FGFR2b_Y734F mutant- or SH3BP4-depleted cells switches the receptor endocytic route to degradation, resulting in decreased breast cancer cell migration and the inhibition of epithelial branching in mouse lung explants. Altogether, these results identify an intriguing ligand-dependent mechanism for the control of receptor fate and cellular outputs that may explain the pathogenic role of deregulated FGFR2b, thus offering therapeutic opportunities.

PICH: a DNA translocase specially adapted for processing anaphase bridge DNA.

The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including the Bloom's syndrome protein (BLM). However, very little is known about the function of PICH or how it is recruited to UFBs. Using a combination of microfluidics, fluorescence microscopy, and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to duplex DNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding DNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a model in which PICH recognizes and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids.

Self-assembly of designed coiled coil peptides studied by small-angle X-ray scattering and analytical ultracentrifugation.

α-Helical coiled coil structures, which are noncovalently associated heptad repeat peptide sequences, are ubiquitous in nature. Similar amphipathic repeat sequences have also been found in helix-containing proteins and have played a central role in de novo design of proteins. In addition, they are promising tools for the construction of nanomaterials. Small-angle X-ray scattering (SAXS) has emerged as a new biophysical technique for elucidation of protein topology. Here, we describe a systematic study of the self-assembly of a small ensemble of coiled coil sequences using SAXS and analytical ultracentrifugation (AUC), which was correlated with molecular dynamics simulations. Our results show that even minor sequence changes have an effect on the folding topology and the self-assembly and that these differences can be observed by a combination of AUC, SAXS, and circular dichroism spectroscopy. A small difference in these methods was observed, as SAXS for one peptide and revealed the presence of a population of longer aggregates, which was not observed by AUC.

Direct binding between BubR1 and B56-PP2A phosphatase complexes regulate mitotic progression.

BubR1 is a central component of the spindle assembly checkpoint that inhibits progression into anaphase in response to improper kinetochore-microtubule interactions. In addition, BubR1 also helps stabilize kinetochore-microtubule interactions by counteracting the Aurora B kinase but the mechanism behind this is not clear. Here we show that BubR1 directly binds to the B56 family of protein phosphatase 2A (PP2A) regulatory subunits through a conserved motif that is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and polo-like kinase 1 (Plk1). Two highly conserved hydrophobic residues surrounding the serine 670 Cdk1 phosphorylation site are required for B56 binding. Mutation of these residues prevents the establishment of a proper metaphase plate and delays cells in mitosis. Furthermore, we show that phosphorylation of serines 670 and 676 stimulates the binding of B56 to BubR1 and that BubR1 targets a pool of B56 to kinetochores. Our data suggest that BubR1 counteracts Aurora B kinase activity at improperly attached kinetochores by recruiting B56-PP2A phosphatase complexes.

Thermodynamic and kinetic analysis of peptides derived from CapZ, NDR, p53, HDM2, and HDM4 binding to human S100B.

S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2, and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, we screened these peptides against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone (1)H and (15)N resonances were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic K(on) rate constants observed in this study (≥1 × 10(7) M(-1) s(-1)) suggest that S100B utilizes a "fly casting mechanism" in the recognition of these peptide targets.

Thermodynamics of the Trp-cage miniprotein unfolding in urea.

The thermodynamic properties of unfolding of the Trp-cage mini protein in the presence of various concentrations of urea have been characterized using temperature-induced unfolding monitored by far-UV circular dichroism spectroscopy. Analysis of the data using a two-state model allowed the calculation of the Gibbs energy of unfolding at 25 degrees C as a function of urea concentration. This in turn was analyzed by the linear extrapolation model that yielded the dependence of Gibbs energy on urea concentration, i.e. the m-value for Trp-cage unfolding. The m-value obtained from the experimental data, as well as the experimental heat capacity change upon unfolding, were correlated with the structural parameters derived from the three dimensional structure of Trp-cage. It is shown that the m-value can be predicted well using a transfer model, while the heat capacity changes are in very good agreement with the empirical models based on model compounds studies. These results provide direct evidence that Trp-cage, despite its small size, is an excellent model for studies of protein unfolding and provide thermodynamic data that can be used to compare with atomistic computer simulations.

Annexin I and annexin II N-terminal peptides binding to S100 protein family members: specificity and thermodynamic characterization.

The S100 proteins make up a family of dimeric calcium binding proteins that function in response to changing calcium levels. Several S100 binding proteins have been identified; however, the exact biological functions of the S100 proteins are largely unknown as there are several factors which modulate their functions. To address these issues, the specificity of binding of representative members of the human S100 proteins to short N-terminal peptides of annexin I (AnI) and annexin II (AnII) was investigated under controlled experimental conditions. AnI and AnII have been shown previously to interact with S100A11 and S100A10, respectively. This provided a unique opportunity to determine their binding specificity with the other members of the human S100 protein family. It was found that AnI binds S100A6 or S100A11 while AnII binds S100A10 or S100A11. This is the first report of the interaction between S100A6 and AnI. The fact that AnI and AnII bind to selected members of the S100 protein family shows that these interactions are specific and that the mode of binding is different from that of calmodulin, as it was found not to bind AnI or AnII. From the analysis of the thermodynamics of interactions, the binding seems to be entropically driven. It was found that both AnI and AnII undergo a coil-to-helix transition upon binding to their respective binding partners. The observation that there is an overlap in functionality is not surprising due to considerable sequence homology between S100 protein family members. In fact, the functional overlap can explain previous failures of S100 knockout constructs to show any detectable changes in phenotype despite numerous implications of these proteins in important cellular processes.

Unfolding thermodynamics of Trp-cage, a 20 residue miniprotein, studied by differential scanning calorimetry and circular dichroism spectroscopy.

Small proteins provide convenient models for computational studies of protein folding and stability, which are usually compared with experimental data. Until recently, the unfolding of Trp-cage was considered to be a two-state process. However, no direct experimental evidence for this has been presented, and in some cases, the contrary has been suggested. To elucidate a detailed unfolding mechanism, we studied the thermodynamics of unfolding of Trp-cage by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. The observation that at low temperatures only approximately 90-95% of Trp-cage exists in the native conformation presented an analytical challenge. Nevertheless, it was found that the DSC and CD data can be fitted simultaneously to the same set of thermodynamic parameters. The major uncertainty in such a global fit is the heat capacity change upon unfolding, DeltaCp. This can be circumvented by obtaining DeltaCp directly from the difference between heat capacity functions of the native and unfolded states. Using such an analysis it is shown that Trp-cage unfolding can be represented by a two-state model with the following thermodynamic parameters: Tm = 43.9 +/- 0.8 degrees C, DeltaH(Tm) = 56 +/- 2 kJ/mol, DeltaCp = 0.3 +/- 0.1 kJ/(mol.K). Using these thermodynamic parameters it is estimated that Trp-cage is marginally stable at 25 degrees C, DeltaG(25 degrees C) = 3.2 +/- 0.2 kJ/mol, which is only 30% more than the thermal fluctuation energy at this temperature.

Advances in the analysis of conformational transitions in peptides using differential scanning calorimetry.

Differential scanning calorimetry can measure the heat capacity of a protein/peptide solution over a range of temperatures at constant pressure, which is used to determine the enthalpy function of the system. There are several experimental factors that can have a significant impact on the determined enthalpy and subsequent derived thermodynamic parameters. These factors are discussed in terms of sample and instrument preparation, as well as data collection and analysis.

Calorimetric evidence for a two-state unfolding of the beta-hairpin peptide trpzip4.

beta-Sheets are a common secondary structural element found in proteins. The difficulty in studying beta-sheet folding and stability is that their formation is often dependent on the tertiary structural environment within the protein. However, the discovery of water-soluble beta-hairpin peptides has allowed them to be used as model systems because they represent the smallest units of beta-sheet structure independent of tertiary structural context. Trpzip4 has been used as a model beta-hairpin peptide to study beta-hairpin folding and stability because it is highly soluble in aqueous solutions, maintains its monomeric state, and shows reversible cooperative thermal unfolding. The previously determined thermodynamic parameters for trpzip4 thermal unfolding vary depending on the spectroscopic probe used, which questions the assumption that trpzip4 unfolds in a two-state manner. Here we provide direct calorimetric evidence that the unfolding of trpzip4 follows a two-state unfolding mode. Furthermore, the thermal unfolding of trpzip4 monitored using near- and far-UV-CD yielded thermodynamic parameters similar to those determined calorimetrically, providing additional evidence for a two-state unfolding mode.