RESUMEN
Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.
Asunto(s)
Anticuerpos Monoclonales , Quimiometría , Humanos , Estabilidad Proteica , Anticuerpos Monoclonales/química , Desplegamiento Proteico , Conformación Proteica , Estabilidad de MedicamentosRESUMEN
Using light scattering (LS), small-angle X-ray scattering (SAXS), and coarse-grained Monte Carlo (MC) simulations, we studied the self-interactions of two monoclonal antibodies (mAbs), PPI03 and PPI13. With LS measurements, we obtained the osmotic second virial coefficient, B22, and the molecular weight, Mw, of the two mAbs, while with SAXS measurements, we studied the mAbs' self-interaction behavior in the high protein concentration regime up to 125 g/L. Through SAXS-derived coarse-grained representations of the mAbs, we performed MC simulations with either a one-protein or a two-protein model to predict B22. By comparing simulation and experimental results, we validated our models and obtained insights into the mAbs' self-interaction properties, highlighting the role of both ion binding and charged patches on the mAb surfaces. Our models provide useful information about mAbs' self-interaction properties and can assist the screening of conditions driving to colloidal stability.
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Anticuerpos Monoclonales , Anticuerpos Monoclonales/química , Método de Montecarlo , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Rayos XRESUMEN
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.
Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Proteómica , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Movimiento Celular , Ligandos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteolisis , Tirosina/metabolismoRESUMEN
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.
Asunto(s)
Anafase/genética , ADN Helicasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cromátides/metabolismo , ADN Helicasas/química , ADN Helicasas/genética , Humanos , Microscopía Fluorescente/métodos , Ácidos Nucleicos Heterodúplex/metabolismo , Nucleosomas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Mutación Puntual/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Biofisica/métodos , Rastreo Diferencial de Calorimetría/métodos , Dispersión Dinámica de Luz/métodos , Concentración de Iones de Hidrógeno , Péptidos/química , Péptidos/genética , Agregado de Proteínas/genética , Estabilidad Proteica/efectos de los fármacosRESUMEN
Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.
Asunto(s)
Anticuerpos Monoclonales/química , Descubrimiento de Drogas/métodos , Inmunoglobulina G/química , Interferón alfa-2/química , Desplegamiento Proteico , Albúmina Sérica Humana/química , Transferrina/química , Secuencia de Aminoácidos , Almacenaje de Medicamentos , Humanos , Concentración de Iones de Hidrógeno , Concentración Osmolar , Agregado de Proteínas , Estabilidad Proteica , Proyectos de Investigación , SolubilidadRESUMEN
PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain.
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Secuencias de Aminoácidos , ADN Helicasas/química , Dominios Proteicos , Proteínas Represoras/química , Secuencia de Aminoácidos , Sitios de Unión/genética , Cristalografía por Rayos X , ADN Helicasas/genética , ADN Helicasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitosis/genética , Modelos Moleculares , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a unique epitope for SUMO interaction spatially opposite to that observed for canonical SUMO interaction motifs (SIMs). HADDOCK docking simulations using chemical shift perturbations and residual dipolar couplings was employed to obtain a structural model for the ZZ domain-SUMO1 complex. Isothermal titration calorimetry experiments support this model by showing that the mutation of key residues in the binding site abolishes binding and that SUMO1 can simultaneously and non-cooperatively bind both the ZZ domain and a canonical SIM motif. The binding dynamics of SUMO1 was further characterized using (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersions, which define the off rates for the ZZ domain and SIM motif and show that the dynamic binding process has different characteristics for the two cases. Furthermore, in the absence of bound ligands SUMO1 transiently samples a high energy conformation, which might be involved in ligand binding.
Asunto(s)
Proteína de Unión a CREB/química , Epítopos/química , Dominios Proteicos , Proteína SUMO-1/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión/genética , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Calorimetría/métodos , Epítopos/genética , Epítopos/metabolismo , Humanos , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Unión Proteica , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , TermodinámicaRESUMEN
Acquired protection from Plasmodium falciparum malaria takes years to develop, probably reflecting the ability of the parasites to evade immunity. A recent example of this is the binding of the Fc region of IgM to VAR2CSA-type PfEMP1. This interferes with specific IgG recognition and phagocytosis of opsonized infected erythrocytes (IEs) without compromising the placental IE adhesion mediated by this PfEMP1 type. IgM also binds via Fc to several other PfEMP1 proteins, where it has been proposed to facilitate rosetting (binding of uninfected erythrocytes to a central IE). To further dissect the functional role of Fc -mediated IgM binding to PfEMP1, we studied the PfEMP1 protein HB3VAR06, which mediates rosetting and binds IgM. Binding of IgM to this PfEMP1 involved the Fc domains Cµ3-Cµ4 in IgM and the penultimate DBL domain (DBLζ2) at the C-terminus of HB3VAR06. However, IgM binding did not inhibit specific IgG labelling of HB3VAR06 or shield IgG-opsonized IEs from phagocytosis. Instead, IgM was required for rosetting, and each pentameric IgM molecule could bind two HB3VAR06 molecules. Together, our data indicate that the primary function of Fc -mediated IgM binding in rosetting is not to shield IE from specific IgG recognition and phagocytosis as in VAR2CSA-type PfEMP1. Rather, the function appears to be strengthening of IE-erythrocyte interactions. In conclusion, our study provides new evidence on the molecular details and functional significance of rosetting, a long-recognized marker of parasites that cause severe P. falciparum malaria.
Asunto(s)
Anticuerpos Antiprotozoarios/metabolismo , Antígenos de Protozoos/metabolismo , Eritrocitos/parasitología , Inmunoglobulina M/metabolismo , Plasmodium falciparum/inmunología , Proteínas Protozoarias/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas , Unión ProteicaRESUMEN
The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying entry into anaphase until all sister chromatids have become bi-oriented. A key component of the SAC is the Mad2 protein, which can adopt either an inactive open (O-Mad2) or active closed (C-Mad2) conformation. The conversion of O-Mad2 into C-Mad2 at unattached kinetochores is thought to be a key step in activating the SAC. The "template model" proposes that this is achieved by the recruitment of soluble O-Mad2 to C-Mad2 bound at kinetochores through its interaction with Mad1. Whether Mad1 has additional roles in the SAC beyond recruitment of C-Mad2 to kinetochores has not yet been addressed. Here, we show that Mad1 is required for mitotic arrest even when C-Mad2 is artificially recruited to kinetochores, indicating that it has indeed an additional function in promoting the checkpoint. The C-terminal globular domain of Mad1 and conserved residues in this region are required for this unexpected function of Mad1.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Mad2/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/química , Células HeLa , Humanos , Proteínas Nucleares/química , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/metabolismo , Factores de Virulencia/química , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Infecciones Estreptocócicas/metabolismo , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidad , Virulencia , Factores de Virulencia/genéticaRESUMEN
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.
Asunto(s)
Mitosis/fisiología , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatografía en Gel , Células HeLa , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Mitosis/genética , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Técnicas del Sistema de Dos Híbridos , Quinasa Tipo Polo 1RESUMEN
De novo design and chemical synthesis of proteins and of other artificial structures that mimic them is a central strategy for understanding protein folding and for accessing proteins with new functions. We have previously described carbohydrates that act as templates for the assembly of artificial proteins, so-called carboproteins. The hypothesis is that the template preorganizes the secondary structure elements and directs the formation of a tertiary structure, thus achieving structural economy in the combination of peptide, linker, and template. We speculate that the structural information from the template could facilitate protein folding. Here we report the design and synthesis of three-helix-bundle carboproteins on deoxyhexopyranosides. The carboproteins were analyzed by CD, analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and NMR spectroscopy, and this revealed the formation of the first compact and folded monomeric carboprotein, distinctly different from a molten globule. En route to this carboprotein we observed a clear effect originating from the template on protein folding.
RESUMEN
Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2.
Asunto(s)
Expresión Génica , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Células HEK293 , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/aislamiento & purificación , Factor I del Crecimiento Similar a la Insulina/química , Factor II del Crecimiento Similar a la Insulina/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Péptidos/química , Estructura Secundaria de Proteína , Secuencias Repetitivas de Aminoácido , Ultracentrifugación , Dicroismo Circular , Modelos Moleculares , Péptidos/síntesis química , Pliegue de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Espectroscopía de Absorción de Rayos X , Rayos XRESUMEN
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.
Asunto(s)
Factores de Crecimiento Nervioso/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas S100/metabolismo , Secuencia de Aminoácidos , Proteína CapZ/química , Proteína CapZ/metabolismo , Proteínas de Ciclo Celular , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/química , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/química , Termodinámica , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
There are many fluorescence-based applications that can be used to characterize molecular interactions. However, available methods often depend on site-specific labeling techniques or binding-induced changes in conformation or size of the probed target molecule. To overcome these limitations, we applied a ratiometric dual-emission approach that quantifies ligand-induced spectral shifts with sub-nanometer sensitivity. The use of environment-sensitive near-infrared dyes with the method we describe enables affinity measurements and thermodynamic characterization without the explicit need for site-specific labeling or ligand-induced conformational changes. We demonstrate that in-solution spectral shift measurements enable precise characterization of molecular interactions for a variety of biomolecules, including proteins, antibodies, and nucleic acids. Thereby, the described method is not limited to a subset of molecules since even the most challenging samples of research and drug discovery projects like membrane proteins and intrinsically disordered proteins can be analyzed.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/metabolismo , Ligandos , Conformación Molecular , Espectrometría de Fluorescencia/métodos , TermodinámicaRESUMEN
High throughput screening for measuring the stability of industrially relevant proteins and their variants is necessary for quality assessment in the development process. Advances in automation, measurement time and sample consumption for many techniques allow rapid measurements with minimal amount of protein. However, many methods include automated data analysis, potentially neglecting important aspects of the protein's behavior in certain conditions. In this study we implement small angle X-ray scattering (SAXS), typically not used to assess protein behavior in industrial screening, in a high throughput screening workflow to address problems of contradicting results and reproducibility among different high throughput methods. As a case study we use the lipases of Thermomyces lanuginosus and Rhizomucor miehei, widely used industrial biocatalysts. We show that even the initial analysis of the SAXS data without performing any time-consuming modelling provide valuable information on interparticle interactions. We conclude that recent advances in automation and data processing, have enabled SAXS to be used more widely as a tool to gain in-depth knowledge highly useful for protein formulation development. This is especially relevant in light of increasing accessibility to SAXS due to the commercial availability of benchtop instruments.
Asunto(s)
Estabilidad Proteica , Proteínas/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Reproducibilidad de los Resultados , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
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.
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Péptidos , Fluorescencia , Cinética , Preparaciones Farmacéuticas , TermodinámicaRESUMEN
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.