RESUMEN
Patients with type 1 diabetes mellitus who are dependent on an external supply of insulin develop insulin-derived amyloidosis at the sites of insulin injection. A major component of these plaques is identified as full-length insulin consisting of the two chains A and B. While there have been several reports that characterize insulin misfolding and the biophysical properties of the fibrils, atomic-level information on the insulin fibril architecture remains elusive. We present here an atomic resolution structure of a monomorphic insulin amyloid fibril that has been determined using magic angle spinning solid-state NMR spectroscopy. The structure of the insulin monomer yields a U-shaped fold in which the two chains A and B are arranged in parallel to each other and are oriented perpendicular to the fibril axis. Each chain contains two ß-strands. We identify two hydrophobic clusters that together with the three preserved disulfide bridges define the amyloid core structure. The surface of the monomeric amyloid unit cell is hydrophobic implicating a potential dimerization and oligomerization interface for the assembly of several protofilaments in the mature fibril. The structure provides a starting point for the development of drugs that bind to the fibril surface and disrupt secondary nucleation as well as for other therapeutic approaches to attenuate insulin aggregation.
Asunto(s)
Amiloide , Insulina , Humanos , Amiloide/química , Amiloide/metabolismo , Insulina/química , Insulina/metabolismo , Modelos Moleculares , Interacciones Hidrofóbicas e Hidrofílicas , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Conformación Proteica , Espectroscopía de Resonancia MagnéticaRESUMEN
In this work, the effect of the salt modulators potassium chloride, ammonium chloride, ammonium sulfate, and potassium sulfate on the elution behavior of insulin in reversed-phase chromatography with ethanol as the organic modifier was investigated. Without the addition of salt modulators, insulin shows the formation of multiple peaks under non-linear loading conditions, presumably due to an aggregate formation equilibrium. Flow rate and temperature did not influence the appearance of multiple peaks. The addition of chloride and sulfate salt modulators changed the monomer-multimer equilibrium, and multi-peak formation no longer occurred. Chloride salts induce a Langmuirian elution behavior, whereas sulfate salts induce additional insulin-insulin interactions resulting in an anti-Langmuirian elution behavior. The elution behavior can be influenced by the combination of both chloride and sulfate salts and by varying the concentration ratio. The separation with respect to two product-related impurities also showed significant differences under Langmuirian and anti-Langmuirian elution conditions and the purification of insulin could be optimized. Induced anti-Langmuirian elution by lowering the chloride/sulfate ratio suppresses an observed tag-along effect of one variant resulting in a slightly smaller pool volume with increased insulin concentration and a significantly increased insulin recovery.
Asunto(s)
Insulina , Sales (Química) , Sales (Química)/química , Cromatografía de Fase Inversa , Cloruros , Cloruro de Amonio , Cloruro de Sodio/química , SulfatosRESUMEN
In process development and characterization, the scale-up of chromatographic steps is a crucial part and brings a number of challenges. Usually, scale-down models are used to represent the process step, and constant column properties are assumed. The scaling is then typically based on the concept of linear scale-up. In this work, a mechanistic model describing an anti-Langmuirian to Langmuirian elution behavior of a polypeptide, calibrated with a pre-packed 1 ml column, is applied to demonstrate the scalability to larger column volumes up to 28.2 ml. Using individual column parameters for each column size, scaling to similar eluting salt concentrations, peak heights, and shapes is experimentally demonstrated by considering the model's relationship between the normalized gradient slope and the eluting salt concentration. Further scale-up simulations show improved model predictions when radial inhomogeneities in packing quality are considered.
RESUMEN
The mechanistic modeling of preparative liquid chromatography is still a challenging task. Nonideal thermodynamic conditions may require activity coefficients for the mechanistic description of preparative chromatography. In this work, a chromatographic cation exchange step with a polypeptide having a complex elution behavior in low and high loading situations is modeled. Model calibration in the linear range of the isotherm is done by applying counterion-induced linear gradient elution experiments between pH 3.3 and 4.3. Inverse fitting with column loads up to 25 mg/mLCV is performed for parameter estimation in the nonlinear range. The polypeptide elution peak shows an anti-Langmuirian behavior with fronting under low loading conditions and a switch to a Langmuirian behavior with increasing load. This unusual elution behavior could be described with an extended version of the sigmoidal Self-Association isotherm including two activity coefficients for the polypeptide and counterion in solution. The activity coefficient of the solute polypeptide shows a strong influence on the model parameters and is crucial in the linear and nonlinear range of the isotherm. The modeling procedure results in a unique and robust model parameter set that is sufficient to describe the complex elution behavior and allows modeling over the full isotherm range.
Asunto(s)
Péptidos , Calibración , Cationes , Cromatografía por Intercambio Iónico/métodos , Cromatografía Liquida/métodos , TermodinámicaRESUMEN
Botulinum neurotoxins (BoNTs) are the most potent toxic proteins to mankind known but applied in low doses trigger a localized muscle paralysis that is beneficial for the therapy of several neurological disorders and aesthetic treatment. The paralytic effect is generated by the enzymatic activity of the light chain (LC) that cleaves specifically one of the SNARE proteins responsible for neurotransmitter exocytosis. The activity of the LC in a BoNT-containing therapeutic can be compromised by denaturing agents present during manufacturing and/or in the cell. Stabilization of the LC by reducing vulnerability towards denaturants would thus be advantageous for the development of BoNT-based therapeutics. In this work, we focused on increasing the stability of LC of BoNT/A1 (LC/A1) towards oxidative stress. We tackled this task by rational design of mutations at cysteine and methionine LC/A1 sites. Designed mutants showed improved oxidative stability in vitro and equipotency to wildtype toxin in vivo. Our results suggest that suitable modification of the catalytic domain can lead to more stable BoNTs without impairing their therapeutic efficacy.
Asunto(s)
Toxinas Botulínicas/genética , Toxinas Botulínicas/toxicidad , Clostridioides difficile/metabolismo , Animales , Toxinas Botulínicas/química , Toxinas Botulínicas/metabolismo , Clostridioides difficile/genética , Ratones , Modelos Moleculares , Actividad Motora/efectos de los fármacos , Mutación , Estrés Oxidativo , Parálisis/inducido químicamente , Conformación ProteicaRESUMEN
Botulinum neurotoxins (BoNTs) are classified by their antigenic properties into seven serotypes (A-G) and in addition by their corresponding subtypes. They are further characterized by divergent onset and duration of effect. Injections of low doses of botulinum neurotoxins cause localized muscle paralysis that is beneficial for the treatment of several medical disorders and aesthetic indications. Optimizing the therapeutic properties could offer new treatment opportunities. This report describes a rational design approach to modify the pharmacological properties by mutations in the C-terminus of BoNT/A1 light chain (LC). Toxins with C-terminal modified LC's displayed an altered onset and duration of the paralytic effect in vivo. The level of effect was dependent on the kind of the mutation in the sequence of the C-terminus. A mutant with three mutations (T420E F423M Y426F) revealed a faster onset and a shorter duration than BoNT/A1 wild type (WT). It could be shown that the C-terminus of BoNT/A1-Lc controls both onset and duration of effect. Thus, it is possible to create a mutated BoNT/A1 with different pharmacological properties which might be useful in the therapy of new indications. This strategy opens the way to design BoNT variants with novel and useful properties.
Asunto(s)
Toxinas Botulínicas Tipo A/farmacología , Diseño de Fármacos , Neurotoxinas/farmacología , Animales , Toxinas Botulínicas Tipo A/farmacocinética , Escherichia coli , Inyecciones Intramusculares , Ratones , Músculo Esquelético/efectos de los fármacos , Neurotoxinas/farmacocinética , Parálisis/inducido químicamente , Relación Estructura-Actividad , Factores de TiempoRESUMEN
In addition to the traditional 1-butanol production by hydroformylation of gaseous propene and by fermentation of biomass, the cytochrome P450-catalyzed direct terminal oxidation of n-butane into the primary alcohol 1-butanol constitutes an alternative route to provide the high demand of this basic chemical. Moreover the use of n-butane offers an unexploited ubiquitous feed stock available in large quantities. Based on protein engineering of CYP153A from Polaromonas sp. JS666 and the improvement of the native redox system, a highly ω-regioselective (>96%) fusion protein variant (CYP153AP.sp.(G254A)-CPRBM3) for the conversion of n-butane into 1-butanol was developed. Maximum yield of 3.12g/L butanol, of which 2.99g/L comprise for 1-butanol, has been obtained after 20h reaction time. Due to the poor solubility of n-butane in an aqueous system, a high pressure reaction assembly was applied to increase the conversion. After optimization a maximum product content of 4.35g/L 1-butanol from a total amount of 4.53g/L butanol catalyzed by the self-sufficient fusion monooxygenase has been obtained at 15bar pressure. In comparison to the CYP153A wild type the 1-butanol concentration was enhanced fivefold using the engineered monooxygenase whole cell system by using the high-pressure reaction assembly.
Asunto(s)
1-Butanol/síntesis química , Butanos/síntesis química , Sistema Enzimático del Citocromo P-450/química , Ingeniería de Proteínas , 1-Butanol/química , Butanos/química , Sistema Enzimático del Citocromo P-450/genética , Fermentación , Oxidación-Reducción , PresiónRESUMEN
A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153AM. aq. -CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments yielded 1.2 g l(-1) ω-hydroxy dodecanoic from 10 g l(-1) C12-FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l(-1) ω-hydroxy dodecanoic acid. We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Láuricos/metabolismo , Alteromonadaceae/enzimología , Alteromonadaceae/genética , Bacillus megaterium/enzimología , Bacillus megaterium/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Hidroxilación , Ácidos Láuricos/química , Ingeniería Metabólica , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
CYP153A from Marinobacter aquaeolei has been identified as a fatty acid ω-hydroxylase with a broad substrate range. Two hotspots predicted to influence substrate specificity and selectivity were exchanged. Mutant G307A is 2- to 20-fold more active towards fatty acids than the wild-type. Residue L354 is determinant for the enzyme ω-regioselectivity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Grasos/biosíntesis , Oxigenasas de Función Mixta/metabolismo , Proteínas Bacterianas/genética , Ácidos Grasos/química , Hidroxilación , Marinobacter/enzimología , Oxigenasas de Función Mixta/genética , Mutación , Estereoisomerismo , Especificidad por SustratoRESUMEN
The oxofunctionalization of saturated hydrocarbons is an important goal in basic and applied chemistry. Biocatalysts like cytochrome P450 enzymes can introduce oxygen into a wide variety of molecules in a very selective manner, which can be used for the synthesis of fine and bulk chemicals. Cytochrome P450 enzymes from the CYP153A subfamily have been described as alkane hydroxylases with high terminal regioselectivity. Here we report the product yields resulting from C(5)-C(12) alkane and alcohol oxidation catalyzed by CYP153A enzymes from Mycobacterium marinum (CYP153A16) and Polaromonas sp. (CYP153A P. sp.). For all reactions, byproduct formation is described in detail. Following cloning and expression in Escherichia coli, the activity of the purified monooxygenases was reconstituted with putidaredoxin (CamA) and putidaredoxin reductase (CamB). Although both enzyme systems yielded primary alcohols and α,ω-alkanediols, each one displayed a different oxidation pattern towards alkanes. For CYP153A P. sp. a predominant ω-hydroxylation activity was observed, while CYP153A16 possessed the ability to catalyze both ω-hydroxylation and α,ω-dihydroxylation reactions.