RESUMO
Since the first administration of insulin to a person with diabetes in 1922, scientific contributions from academia and industry have improved insulin therapy and access. The pharmaceutical need for insulin is now more than 40 tons annually, half of which is produced by recombinant secretory expression in Saccharomyces cerevisiae. We discuss how, in this yeast species, adaptation of insulin precursors by removable structural elements is pivotal for efficient secretory expression. The technologies reviewed have been implemented at industrial scale and are seminal for the supply of human insulin and insulin analogues to people with diabetes now and in the future. Engineering of a target protein with removable structural elements may provide a general approach to yield optimisation.
Assuntos
Diabetes Mellitus , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Insulina/genética , Proteínas Recombinantes/metabolismoRESUMO
Insulin icodec is a once-weekly insulin analogue that has a long half-life of approximately 7 days, making it suitable for once weekly dosing. The Insulin icodec molecule was developed based on the hypothesis that lowering insulin receptor affinity and introducing a strong albumin-binding moiety would result in a long insulin half-life, provided that non-receptor-mediated clearance is diminished. Here, we report an insulin clearance mechanism, resulting in the splitting of insulin molecules into its A-chain and B-chain by a thiol-disulphide exchange reaction. Even though the substitutions in insulin icodec significantly stabilise insulin against such degradation, some free B-chain is observed in plasma samples from minipigs and people with type 2 diabetes. In summary, we identify thiol-disulphide exchange reactions to be an important insulin clearance mechanism and find that stabilising insulin icodec towards this reaction significantly contributes to its long pharmacokinetic/pharmacodynamic profile.
Assuntos
Diabetes Mellitus Tipo 2 , Dissulfetos , Insulina , Animais , Humanos , Masculino , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/sangue , Dissulfetos/química , Meia-Vida , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/química , Insulina/administração & dosagem , Insulina/metabolismo , Insulina/química , Insulina/farmacocinética , Receptor de Insulina/metabolismo , Compostos de Sulfidrila/química , Suínos , Porco MiniaturaRESUMO
Inherited bleeding disorders such as Glanzmann thrombasthenia (GT) lack prophylactic treatment options. As a result, serious bleeding episodes are treated acutely with blood product transfusions or frequent, repeated intravenous administration of recombinant activated coagulation factor VII (rFVIIa). Here we describe HMB-001, a bispecific antibody designed to bind and accumulate endogenous FVIIa and deliver it to sites of vascular injury by targeting it to the TREM (triggering receptor expressed on myeloid cells)-like transcript-1 (TLT-1) receptor that is selectively expressed on activated platelets. In healthy nonhuman primates, HMB-001 prolonged the half-life of endogenous FVIIa, resulting in its accumulation. Mouse bleeding studies confirmed antibody-mediated potentiation of FVIIa hemostatic activity by TLT-1 targeting. In ex vivo models of GT, HMB-001 localized FVIIa on activated platelets and potentiated fibrin-dependent platelet aggregation. Taken together, these results indicate that HMB-001 has the potential to offer subcutaneous prophylactic treatment to prevent bleeds in people with GT and other inherited bleeding disorders, with a low-frequency dosing regimen.
Assuntos
Anticorpos Biespecíficos , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/imunologia , Humanos , Fator VIIa , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Camundongos , Modelos Animais de Doenças , Hemorragia/prevenção & controle , Hemorragia/tratamento farmacológico , Agregação Plaquetária/efeitos dos fármacos , Trombastenia/tratamento farmacológico , Trombastenia/imunologia , Camundongos Endogâmicos C57BL , Feminino , Masculino , Macaca fascicularis , Ativação Plaquetária/efeitos dos fármacosRESUMO
Insulin degludec, an engineered acylated insulin, was recently reported to form a soluble depot after subcutaneous injection with a subsequent slow release of insulin and an ultralong glucose-lowering effect in excess of 40 h in humans. We describe the structure, ligand binding properties, and self-assemblies of insulin degludec using orthogonal structural methods. The protein fold adopted by insulin degludec is very similar to that of human insulin. Hexamers in the R(6) state similar to those of human insulin are observed for insulin degludec in the presence of zinc and resorcinol. However, under conditions comparable to the pharmaceutical formulation comprising zinc and phenol, insulin degludec forms finite dihexamers that are composed of hexamers in the T(3)R(3) state that interact to form an R(3)T(3)-T(3)R(3) structure. When the phenolic ligand is depleted and the solvent condition thereby mimics that of the injection site, the quaternary structure changes from dihexamers to a supramolecular structure composed of linear arrays of hundreds of hexamers in the T(6) state and an average molar mass, M(0), of 59.7 × 10(3) kg/mol. This novel concept of self-assemblies of insulin controlled by zinc and phenol provides the basis for the slow action profile of insulin degludec. To the best of our knowledge, this report for the first time describes a tight linkage between quaternary insulin structures of hexamers, dihexamers, and multihexamers and their allosteric state and its origin in the inherent propensity of the insulin hexamer for allosteric half-site reactivity.
Assuntos
Insulina de Ação Prolongada/química , Insulina de Ação Prolongada/metabolismo , Fenol/metabolismo , Zinco/metabolismo , Acetilação , Sequência de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Resorcinóis/metabolismo , Espalhamento a Baixo Ângulo , Ultracentrifugação , Difração de Raios XRESUMO
A series of bovine insulin samples were obtained as 14 polycrystalline precipitates at room temperature in the pH range 5.0-7.6. High-resolution powder X-ray diffraction data were collected to reveal the T6 hexameric insulin form. Sample homogeneity and reproducibility were verified by additional synchrotron measurements using an area detector. Pawley analyses of the powder patterns displayed pH- and radiation-induced anisotropic lattice modifications. The pronounced anisotropic lattice variations observed for T6 insulin were exploited in a 14-data-set Rietveld refinement to obtain an average crystal structure over the pH range investigated. Only the protein atoms of the known structure with PDB code 2a3g were employed in our starting model. A novel approach for refining protein structures using powder diffraction data is presented. In this approach, each amino acid is represented by a flexible rigid body (FRB). The FRB model requires a significantly smaller number of refinable parameters and restraints than a fully free-atom refinement. A total of 1542 stereochemical restraints were imposed in order to refine the positions of 800 protein atoms, two Zn atoms and 44 water molecules in the asymmetric unit using experimental data in the resolution range 18.2-2.7 Å for all profiles.
Assuntos
Insulina Ultralenta/química , Insulina/química , Animais , Anisotropia , Bovinos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Difração de Pó/métodos , Difração de Raios X/métodosRESUMO
The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. Great attention has been put on improved secretory production of active pharmaceutical ingredients, and the secretory pathway of this eukaryotic host has been the playground of diverse strain engineering studies, aiming at enhanced cellular capacities for folding and trafficking of the target proteins. However, the cellular quality assessment for secretory proteins remains mostly unpredictable, and different target proteins often do not picture similar secretion yields, underlining the dependency of efficient secretion on the physicochemical properties of the protein of interest. In this study, two human insulin analog precursors (IAPs) with minor differences in their amino acid sequences were used as model secretory proteins. No differences between cells expressing these two proteins were found in the IAP transcript levels, gene copy numbers, or intra-cellularly accumulated proteins, yet a more than sevenfold difference in their secretion yields was found. Physiological characterization of cells expressing these proteins in batch processes revealed no significant difference in their specific growth rate, but an altered overflow metabolism. Global transcriptome analysis carried out in chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated and indicated an increased degradation of the IAP with the low secretion yield. In silico protein structure modeling of the IAPs suggested a difference in conformational stability, induced by the amino acid substitution, which most likely resulted in disparity in trafficking through the secretory pathway and thus a large difference in secretion yields.
Assuntos
Insulinas/química , Insulinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Reatores Biológicos/microbiologia , Técnicas de Cultura de Células/métodos , Simulação por Computador , Proteínas Fúngicas/análise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Humanos , Insulinas/genética , Modelos Moleculares , Proteólise , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , TranscriptomaRESUMO
We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel ß-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel ß-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.
Assuntos
Sequência Conservada , Drosophila melanogaster , Evolução Molecular , Insulina/química , Insulina/metabolismo , Proteínas/química , Proteínas/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Cristalografia por Raios X , Feminino , Humanos , Insulina/farmacologia , Radioisótopos do Iodo , Lipogênese/efeitos dos fármacos , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas/farmacologia , Ratos , Receptor de Insulina/metabolismo , Trealose/metabolismoRESUMO
The effects of the ligands phenol and resorcinol on the crystallization of human insulin have been investigated as a function of pH. Powder diffraction data were used to characterize several distinct polymorphic forms. A previously unknown polymorph with monoclinic symmetry (P2(1)) was identified for both types of ligand with similar characteristics [the unit-cell parameters for the insulin-resorcinol complex were a = 114.0228 (8), b = 335.43 (3), c = 49.211â (6) Å, ß = 101.531 (8)°].
Assuntos
Insulina/química , Fenol/química , Resorcinóis/química , Cristalização , Humanos , Concentração de Íons de Hidrogênio , Difração de Pó , Conformação ProteicaRESUMO
Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human insulin receptor (hIR) in an active state obtained at non-saturating, physiologically relevant insulin conditions. Insulin binds asymmetrically to the receptor under these conditions, occupying up to three of the four possible binding sites. Deletion analysis of the receptor together with site specific peptides and insulin analogs used in binding studies show that both sites 1 and 2 are required for high insulin affinity. We identify a homotypic interaction of the fibronectin type III domain (FnIII-3) of IR resulting in tight interaction of membrane proximal domains of the active, asymmetric receptor dimer. Our results show how insulin binding at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer. We propose an insulin binding and activation mechanism, which is sequential, exhibits negative cooperativity, and is based on asymmetry at physiological insulin concentrations with one to three insulin molecules activating IR.
Assuntos
Antígenos CD , Insulina , Receptor de Insulina , Antígenos CD/química , Antígenos CD/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Insulina/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Transdução de SinaisRESUMO
Through binding to and signaling via the insulin receptor (IR), insulin is involved in multiple effects on growth and metabolism. The current model for the insulin-IR binding process is one of a biphasic reaction. It is thought that the insulin peptide possesses two binding interfaces (sites 1 and 2), which allow it to bridge the two alpha-subunits of the insulin receptor during the biphasic binding reaction. The sequential order of the binding events involving sites 1 and 2, as well as the molecular interactions corresponding to the fast and slow binding events, is still unknown. In this study we examined the series of events that occur during the binding process with the help of three insulin analogues: insulin, an analogue mutated in site 2 (B17A insulin), and an analogue in which part of site 1 was deleted (Des A1-4 insulin), both with and without a fluorescent probe attached. The binding properties of these analogues were tested using two soluble Midi IR constructs representing the two naturally occurring isoforms of the IR, Midi IR-A and Midi IR-B. Our results showed that in the initial events leading to Midi IR-insulin complex formation, insulin site 2 binds to the IR in a very fast binding event. Subsequent to this initial fast phase, a slower rate-limiting phase occurs, consistent with a conformational change in the insulin-IR complex, which forms the final high-affinity complex. The terminal residues A1-A4 of the insulin A-chain are shown to be important for the slow binding phase, as insulin lacking these amino acids is unable to induce a conformational change of IR and has a severely impaired binding affinity. Moreover, differences in the second phase of the binding process involving insulin site 1 between the IR-A and IR-B isoforms suggest that the additional amino acids encoded by exon 11 in the IR-B isoform influence the binding process.
Assuntos
Insulina/metabolismo , Receptor de Insulina/metabolismo , Sítios de Ligação , Éxons , Humanos , Insulina/química , Insulina/genética , Cinética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptor de Insulina/genéticaRESUMO
In this study, the first crystal structure of a novel crystal form of human insulin bound to meta-cresol in an acidic environment is reported. The combination of single-crystal and powder X-ray diffraction crystallography led to the detection of a previously unknown monoclinic phase (P21). The structure was identified from the powder patterns and was solved using single-crystal diffraction data at 2.2â Å resolution. The unit-cell parameters at pH 6.1 are a = 47.66, b = 70.36, c = 84.75â Å, ß = 105.21°. The structure consists of two insulin hexamers per asymmetric unit. The potential use of this insulin form in microcrystalline drugs is discussed.
Assuntos
Cresóis/química , Insulina/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Difração de Raios XRESUMO
Ultralente insulin has been one of the commercially most important insulin preparations in diabetes treatment over the last 50 years. It is a suspension of insulin microcrystals which dissolve slowly following subcutaneous injection. Because of the small crystal size of about 25 x 25 x 5 microm(3) the atomic structure has been elusive until now. Here we present the crystal structures from Ultralente and their precursor microcrystals from the industrial manufacturing process. During this process insulin undergoes a conformational change within the microcrystals. Both structures show canonical folding of the insulin molecules but exhibit a number of new features when compared with other insulin structures. Surprisingly, we found that the Ultralente crystals bind the conservation agent methylparaben, which slows down dissolution of the crystals and thus contributes to the long duration of action.
Assuntos
Hipoglicemiantes/química , Insulina de Ação Prolongada/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Parabenos/química , Parabenos/metabolismo , Conformação ProteicaRESUMO
Translation across species of immunoassay results is often challenging due to the lack of cross-species reactivity of antibodies. In order to investigate the biology of insulin and IGF1 receptors, we generated new versatile monoclonal assay antibodies using the extracellular domain of the insulin/IGF1 hybrid receptor as the bait protein in the Adimab yeast antibody discovery platform and as the antigen in a rabbit monoclonal antibody platform. The resulting antibody clones were screened for receptor specificity as well as cross-species reactivity to both tissue and cell line derived samples. Using these strategies, we were able to identify highly specific insulin receptor monoclonal antibodies that lack cross-reactivity to the IGF1 receptor using the Adimab platform and a highly specific IGF1 receptor monoclonal antibody that lacks cross-reactivity to the insulin receptor using the rabbit antibody platform. Unlike earlier monoclonal antibodies reported in the literature, these antibodies show cross-species reactivity to the extracellular domains of mouse, rat, pig, and human receptors, indicating that they bind conserved epitopes. Furthermore, the antibodies work well in several different assay formats, including ELISA, flow cytometry, and immunoprecipitation, and therefore provide new tools to study insulin and IGF1 receptor biology with translation across several species and experimental model systems.
Assuntos
Anticorpos Monoclonais/imunologia , Receptor IGF Tipo 1/imunologia , Receptor de Insulina/imunologia , Animais , Anticorpos Monoclonais/química , Reações Cruzadas , Células HCT116 , Humanos , Camundongos , Coelhos , Ratos , Especificidade da Espécie , SuínosRESUMO
The insulin/insulin-like growth factor signalling axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth and lifespan in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in blood in complexes with IGF-binding proteins (IGFBP1-6). Insect insulin-like polypeptide binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. Here, we report structures of the Drosophila IBP Imp-L2 in its free form and bound to Drosophila insulin-like peptide 5 and human IGF-1. Imp-L2 contains two immunoglobulin-like fold domains and its architecture is unrelated to human IGFBPs, suggesting a distinct strategy for bioavailability regulation of insulin-like hormones. Similar hormone binding modes may exist in other insect vectors, as the IBP sequences are highly conserved. Therefore, these findings may open research routes towards a rational interference of transmission of diseases such as malaria, dengue and yellow fevers.
Assuntos
Proteínas de Drosophila/química , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/química , Animais , Disponibilidade Biológica , Drosophila , Humanos , Insulina/farmacocinética , Fator de Crescimento Insulin-Like I/química , Conformação ProteicaRESUMO
BACKGROUND: Insulin is a therapeutic protein that is widely used for the treatment of diabetes. Its biological function was discovered more than 80 years ago and it has since then been characterized extensively. Crystallization of the insulin molecule has always been a key activity since the protein is often administered by subcutaneous injections of crystalline insulin formulations. Over the years, insulin has been crystallized and characterized in a number of crystal systems. RESULTS: Interestingly, we have now discovered two new crystal forms of human insulin. The crystals were obtained when the two chaotropic agents, urea and thiocyanate were present in the crystallization experiments, and their structures were determined by X-ray crystallography. The crystals belong to the orthorhombic and monoclinic crystal systems, with space groups C2221 and C2 respectively. The orthorhombic crystals were obtained at pH 6.5 and contained three insulin hexamers in R6 conformation in the asymmetric unit whilst the monoclinic C2 crystals were obtained at pH 7.0 and contained one R6 hexamer in the asymmetric unit. Common for the two new crystals is a hexamer-hexamer interaction that has not been found in any of the previous crystal forms of insulin. The contacts involve a tight glutamate-glutamate interaction with a distance of 2.3 A between groups. The short distance suggests a low barrier hydrogen bond. In addition, two tyrosine-tyrosine interactions occupying a known phenol binding pocket contribute to the stabilization of the contacts. Within the crystals, distinct binding sites for urea were found, adding further to the discussion on the role of urea in protein denaturation. CONCLUSION: The change in space group from C2221 to C2 was primarily caused by an increase in pH. The fewer number of hexamer-hexamer interactions comprising the short hydrogen bond in the C2 space group suggest that pH is the driving force. In addition, the distance between the two glutamates increases from 2.32 A in the C2221 crystals to 2.4 A in the C2 crystals. However, in both cases the low barrier hydrogen bond and the tyrosine-tyrosine interaction should contribute to the stability of the crystals which is crucial when used in pharmaceutical formulations.
Assuntos
Cristalografia por Raios X , Insulina/química , Tiocianatos/química , Ureia/química , Sítios de Ligação , Cristalização/métodos , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica , Tiocianatos/metabolismo , Ureia/metabolismoRESUMO
Insulin NPH (neutral protamine hagedorn) has for long been one of the most important therapeutic formulations for the treatment of diabetes. The protracted action profile of NPH formulations is gained from crystallizing insulin with zinc in the presence of the basic poly-arginine peptide protamine. In spite of its long history and successful use, the binding mode of the insulin-protamine complex is not known. In this study, three different systems were used to study protamine binding to insulin. In the first system, crystals of an insulin-protamine complex grown in the presence of urea and diffracting to 1.5A resolution were analyzed. In the second system, a shorter peptide consisting of 12 arginine residues was co-crystallized with insulin in order to reduce the flexibility and thereby improve the electron density of the peptide. Both systems yielded data to a significantly higher resolution than obtained previously. In addition, a third system was analyzed where crystals of insulin and protamine were grown in the absence of urea, with conditions closely resembling the pharmaceutical formulation. Data from these NPH microcrystals could for the first time be collected to 2.2A resolution at a micro focused X-ray beamline. Analysis of all three crystal forms reveal potential protamine density located close to the solvent channel leading to the centrally located zinc atoms in the insulin hexamer and support that protamine binds to insulin in a not well defined conformation.
Assuntos
Hipoglicemiantes/química , Insulina Isófana/química , Protaminas/química , Zinco/química , Sítios de Ligação , Carbamatos/química , Química Farmacêutica , Cristalização , Cristalografia por Raios X/métodos , Humanos , Hipoglicemiantes/metabolismo , Insulina Isófana/metabolismo , Modelos Moleculares , Estrutura Molecular , Peptídeos/química , Protaminas/metabolismo , Ligação Proteica , Conformação Proteica , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ureia/química , Zinco/metabolismoRESUMO
The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors.
Assuntos
Anticorpos/química , Anticorpos/imunologia , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligação Proteica , Conformação ProteicaRESUMO
The small-angle X-ray scattering (SAXS) methodology enables structural characterization of biological macromolecules in solution. However, because SAXS provides low-dimensional information, several potential structural configurations can reproduce the experimental scattering profile, which severely complicates the structural refinement process. Here, we present a bias-exchange metadynamics refinement protocol that incorporates SAXS data as collective variables and therefore tags all possible configurations with their corresponding free energies, which allows identification of a unique structural solution. The method has been implemented in PLUMED and combined with the GROMACS simulation package, and as a proof of principle, we explore the Trp-cage protein folding landscape.
Assuntos
Proteínas/química , Espalhamento a Baixo Ângulo , Termodinâmica , Difração de Raios X , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de ProteínaRESUMO
Amyloid fibrils share various common structural features and their presence can be detected by Thioflavin T (ThT). In this paper, the binding mode of ThT to insulin amyloid fibrils was examined. Scatchard analysis and isothermal titration calorimetry (ITC) showed at least two binding site populations. The binding site population with the strongest binding was responsible for the characteristic ThT fluorescence. This binding had a capacity of about 0.1 moles of ThT bound per mole of insulin in fibril form. The binding capacity was unaffected by pH, but the affinity was lowest at low pH. Notably, presence of a third binding process prior to the other processes was suggested by ITC. Binding of ThT resulted in only minor changes in the fibril structure according to the X-ray diffraction patterns, where a slightly more dominant equatorial reflection at 16A relative to the intersheet distance of 11A was observed. No change in the interstrand distance of 4.8A was observed. On the basis of our results, we propose that ThT binds in cavities running parallel to the fibril axis, e.g., between the protofilaments forming the fibrils. Such cavities have been proposed previously in insulin fibrils and several other amyloid fibril models.