RESUMEN
There is great interest in identifying a glucagon-like peptide-1 (GLP-1)-based combination therapy that will more effectively promote weight loss in patients with type 2 diabetes. Fibroblast growth factor 21 (FGF21) is a compelling yet previously unexplored drug candidate to combine with GLP-1 due to its thermogenic and insulin-sensitizing effects. Here, we describe the development of a biologic that fuses GLP-1 to FGF21 with an elastin-like polypeptide linker that acts as a sustained release module with zero-order drug release. We show that once-weekly dual-agonist treatment of diabetic mice results in potent weight-reducing effects and enhanced glycemic control that are not observed with either agonist alone. Furthermore, the dual-agonist formulation has superior efficacy compared to a GLP-1/FGF21 mixture, demonstrating the utility of combining two structurally distinct peptides into one multifunctional molecule. We anticipate that these results will spur further investigation into GLP-1/FGF21 multiagonism for the treatment of metabolic disease.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Hiperglucemia , Animales , Preparaciones de Acción Retardada/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Factores de Crecimiento de Fibroblastos/agonistas , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Humanos , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/prevención & control , Ratones , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Péptidos/farmacologíaRESUMEN
Post-traumatic arthritis (PTA) is a rapidly progressive form of arthritis that develops due to joint injury, including articular fracture. Current treatments are limited to surgical restoration and stabilization of the joint; however, evidence suggests that PTA progression is mediated by the upregulation of pro-inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor-α (TNF-α). Although these cytokines provide potential therapeutic targets for PTA, intra-articular injections of anti-cytokine therapies have proven difficult due to rapid clearance from the joint space. In this study, we examined the ability of a cross-linked elastin-like polypeptide (xELP) drug depot to provide sustained intra-articular delivery of IL-1 and TNF-α inhibitors as a beneficial therapy. Mice sustained a closed intra-articular tibial plateau fracture; treatment groups received a single intra-articular injection of drug encapsulated in xELP. Arthritic changes were assessed 4 and 8 weeks after fracture. Inhibition of IL-1 significantly reduced the severity of cartilage degeneration and synovitis. Inhibition of TNF-α alone or with IL-1 led to deleterious effects in bone morphology, articular cartilage degeneration, and synovitis. These findings suggest that IL-1 plays a critical role in the pathogenesis of PTA following articular fracture, and sustained intra-articular cytokine inhibition may provide a therapeutic approach for reducing or preventing joint degeneration following trauma.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Cartílago Articular/efectos de los fármacos , Preparaciones de Acción Retardada/farmacología , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Artritis Experimental/etiología , Artritis Experimental/metabolismo , Proteína de la Matriz Oligomérica del Cartílago/metabolismo , Cartílago Articular/diagnóstico por imagen , Cartílago Articular/lesiones , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/química , Sistemas de Liberación de Medicamentos/métodos , Elastina/química , Inyecciones Intraarticulares , Proteína Antagonista del Receptor de Interleucina 1/administración & dosificación , Proteína Antagonista del Receptor de Interleucina 1/química , Interleucina-1/antagonistas & inhibidores , Interleucina-1/metabolismo , Masculino , Metaloproteinasa 3 de la Matriz/sangre , Metaloproteinasa 3 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/administración & dosificación , Péptidos/química , Líquido Sinovial/efectos de los fármacos , Líquido Sinovial/metabolismo , Temperatura , Factores de Tiempo , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Heridas y Lesiones/complicaciones , Microtomografía por Rayos XRESUMEN
Peptide drugs are an exciting class of pharmaceuticals for the treatment of a variety of diseases; however, their short half-life dictates multiple and frequent injections causing undesirable side effects. Herein, we describe a novel peptide delivery system that seeks to combine the attractive features of prolonged circulation time with a prolonged release formulation. This system consists of glucagon-like peptide-1, a type-2 diabetes drug fused to a thermally responsive, elastin-like-polypeptide (ELP) that undergoes a soluble-insoluble phase transition between room temperature and body temperature, thereby forming an injectable depot. We synthesized a set of GLP-1-ELP fusions and verified their proteolytic stability and potency in vitro. Significantly, a single injection of depot forming GLP-1-ELP fusions reduced blood glucose levels in mice for up to 5 days, 120 times longer than an injection of the native peptide. These findings demonstrate the unique advantages of using ELPs to release peptide-ELP fusions from a depot combined with enhanced systemic circulation to create a tunable peptide delivery system.
Asunto(s)
Glucemia/metabolismo , Preparaciones de Acción Retardada/química , Elastina/administración & dosificación , Péptido 1 Similar al Glucagón/administración & dosificación , Hipoglucemiantes/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Secuencia de Aminoácidos , Animales , Elastina/química , Elastina/farmacología , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/farmacología , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Inyecciones , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Transición de Fase , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Metals support surface plasmons at optical wavelengths and have the ability to localize light to subwavelength regions. The field enhancements that occur in these regions set the ultimate limitations on a wide range of nonlinear and quantum optical phenomena. We found that the dominant limiting factor is not the resistive loss of the metal, but rather the intrinsic nonlocality of its dielectric response. A semiclassical model of the electronic response of a metal places strict bounds on the ultimate field enhancement. To demonstrate the accuracy of this model, we studied optical scattering from gold nanoparticles spaced a few angstroms from a gold film. The bounds derived from the models and experiments impose limitations on all nanophotonic systems.
Asunto(s)
Oro/química , Nanopartículas del Metal/química , Resonancia por Plasmón de Superficie , Hidrodinámica , Luz , Nanosferas/química , Dispersión de RadiaciónRESUMEN
We use optical darkfield micro-spectroscopy to characterize the plasmon resonance of individual silver nanoparticles in the presence of a substrate. The optical system permits multiple individual nanoparticles to be identified visually for simultaneous spectroscopic study. For silver particles bound to a silanated glass substrate, we observe changes in the Plasmon resonance due to induced variations in the local refractive index. The shifts in the plasmon resonance are investigated using a simple analytical theory in which the contributions from the substrate and environment are weighted with distance from the nanoparticle. The theory is compared with experimental results to determine a weighting factor which facilitates modeling of environmental refractive index changes using standard Mie code. Use of the optical system for characterizing nanoparticles attached to substrates for biosensing applications is discussed.
RESUMEN
The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T(t) of an ELP. A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly.
Asunto(s)
Péptidos/química , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Bases de Datos como Asunto , Escherichia coli/metabolismo , Oro Coloide/química , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Plásmidos/metabolismo , Conformación Proteica , Pliegue de Proteína , Homología de Secuencia de Aminoácido , Solubilidad , Resonancia por Plasmón de Superficie , Temperatura , TermodinámicaRESUMEN
Elastin-like polypeptides (ELPs) composed of a VPGXG repeat undergo a reversible phase transition in aqueous solution. They are hydrophilic and soluble in aqueous solution below their transition temperature (T(t)), but they become hydrophobic and aggregate when the temperature is raised above their T(t). In this study, we examine the quantitative uptake of a fluorescence-labeled, thermally responsive ELP as a function of ELP concentration between 5 and 15 microM in solution in response to hyperthermia by three cultured cancer cell lines. Flow cytometry of fluorescein-ELP conjugates showed that hyperthermia enhanced the cellular uptake of the thermally responsive ELP in human ovarian carcinoma cells (SKOV-3) and in HeLa cells at a concentration of 10 microM or higher, but not at a concentration of 5 microM, as compared with the uptake of a thermally inactive ELP control. In FaDu cells, hyperthermia stimulated uptake of the thermally responsive ELP at all solution concentrations of ELP between 5 and 15 microM. In particular, a >2-fold greater uptake of thermally responsive ELP compared with the thermally inactive control ELP was observed for FaDu cells at a solution concentration of 15 microM in heated cells. Confocal fluorescence microscopy of tumor cells incubated with a rhodamine conjugate of the thermally responsive ELP showed that the cytoplasm was uniformly stained below the T(t). Above the T(t), fluorescent particles were observed in the cytoplasm, suggesting that these particles are aggregates of the thermally responsive polypeptide resulting from the ELP phase transition. These studies demonstrate that the endocytotic uptake of a thermally responsive ELP is significantly enhanced by the thermally triggered phase transition of the polypeptide.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Elastina/farmacocinética , Hipertermia Inducida , Neoplasias Ováricas/metabolismo , Péptidos/farmacocinética , Carcinoma de Células Escamosas/terapia , Portadores de Fármacos , Elastina/química , Femenino , Citometría de Flujo , Colorantes Fluorescentes , Calor , Humanos , Cinética , Microscopía Confocal , Neoplasias Ováricas/terapia , Péptidos/química , Fracciones Subcelulares/metabolismo , Células Tumorales CultivadasRESUMEN
We report a new thermal targeting method in which a thermally responsive drug carrier selectively accumulates in a solid tumor that is maintained above physiological temperature by externally applied, focused hyperthermia. We synthesized two thermally responsive polymers that were designed to exhibit a lower critical solution temperature (LCST) transition slightly above physiological temperature: (1) a genetically engineered elastin-like polypeptide (ELP) and (2) a copolymer of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm). The delivery of systemically injected polymer-rhodamine conjugates to solid tumors was investigated by in vivo fluorescence video microscopy of ovarian tumors implanted in dorsal skin fold window chambers in nude mice, with and without local hyperthermia. When tumors were heated to 42 degrees C, the accumulation of a thermally responsive ELP with a LCST of 40 degrees C was approximately twofold greater than the concentration of the same polymer in tumors that were not heated. Similar results were also obtained for a thermally responsive poly(NIPAAM-co-AAm), though the enhanced accumulation of this carrier in heated tumors was lower than that observed for the thermally responsive ELP. These results suggest that enhanced delivery of drugs to solid tumors can be achieved by conjugation to thermally responsive polymers combined with local heating of tumors.
Asunto(s)
Sistemas de Liberación de Medicamentos , Hipertermia Inducida , Polímeros/química , Acrilamidas/química , Animales , Fenómenos Químicos , Química Física , Elastina/química , Ratones , Ratones Desnudos , Microscopía Fluorescente , Microscopía por Video , Nefelometría y Turbidimetría , Espectrofotometría UltravioletaRESUMEN
The change in optical properties of colloidal gold upon aggregation has been used to develop an experimentally convenient colorimetric method to study the interfacial phase transition of an elastin-like polypeptide (ELP), a thermally responsive biopolymer. Gold nanoparticles, functionalized with a self-assembled monolayer (SAM) of mercaptoundecanoic acid onto which an ELP was adsorbed, exhibit a characteristic red color due to the surface plasmon resonance (SPR) of individual colloids. Raising the solution temperature from 10 degrees C to 40 degrees C thermally triggered the hydrophilic-to-hydrophobic phase transition of the adsorbed ELP resulting in formation of large aggregates due to interparticle hydrophobic interaction. Formation of large aggregates caused a change in color of the colloidal suspension from red to violet due to coupling of surface plasmons in aggregated colloids. The surface phase transition of the ELP was reversible, as seen from the reversible change in color upon cooling the suspension to 10 degrees C. The formation of colloidal aggregates due to the interfacial phase transition of adsorbed ELP was independently verified by dynamic light scattering of ELP-modified gold colloids as a function of temperature. Colloidal SPR provides a simple and convenient colorimetric method to study the influence of the solution environment, interfacial properties, and grafting method on the transition properties of ELPs and other environmentally responsive polymers at the solid-water interface.
Asunto(s)
Elastina/química , Oro Coloide/química , Adsorción , Ácidos Grasos/química , Cinética , Tamaño de la Partícula , Soluciones , Espectrofotometría Ultravioleta , Resonancia por Plasmón de Superficie/métodos , Propiedades de Superficie , Agua/químicaRESUMEN
Elastin-like polypeptides (ELPs) undergo a reversible, soluble-to-insoluble phase transition in aqueous solution upon heating through a characteristic transition temperature (T(t)). Incorporating a terminal ELP expression tag into the gene of a protein of interest allows ELP fusion proteins to be purified from cell lysate by cycles of environmentally triggered aggregation, separation from solution by centrifugation, and resolubilization in buffer. In this study, we examine the effect of ELP length on the expression and purification of a thioredoxin-ELP fusion protein and show that reducing the size of the ELP tag from 36 to 9 kDa increases the expression yield of thioredoxin by 4-fold, to a level comparable to that of free thioredoxin expressed without an ELP tag, while still allowing efficient purification. However, truncation of the ELP tag also results in a more complex transition behavior than is observed with larger tags. For both the 36 kDa and the 9 kDa ELP tag fused to thioredoxin, dynamic light scattering showed that large aggregates with hydrodynamic radii of approximately 2 microm form as the temperature is raised to above the T(t). These aggregates persist at all temperatures above the T(t) for the thioredoxin fusion with the 36 kDa ELP tag. With the 9 kDa tag, however, smaller particles with hydrodynamic radii of approximately 12 nm begin to form at the expense of the larger, micron-size aggregates as the temperature is further raised above the T(t). Because only large aggregates can be effectively retrieved by centrifugation, efficient purification of fusion proteins with short ELP tags requires selection of solution conditions that favor the formation of the micron-size aggregates. Despite this additional complexity, our results show that the ELP tag can be successfully truncated to enhance the yield of a target protein without compromising its purification.
Asunto(s)
Elastina/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Tiorredoxinas/aislamiento & purificación , Secuencia de Bases , Centrifugación/métodos , Cromatografía de Afinidad , Elastina/genética , Escherichia coli/genética , Luz , Datos de Secuencia Molecular , Nefelometría y Turbidimetría , Tamaño de la Partícula , Péptidos/genética , Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Dispersión de Radiación , Temperatura , Tiorredoxinas/genéticaRESUMEN
Elastin-like polypeptides (ELPs) are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. They are soluble in aqueous solutions below their transition temperature (T1) but hydrophobically collapse and aggregate at temperatures greater than T1. We hypothesized that ELPs conjugated to drugs would enable thermally targeted drug delivery to solid tumors if their T1 were between body temperature and the temperature in a locally heated region. To test this hypothesis, we synthesized a thermally responsive ELP with a T1 of 41 degrees C and a thermally unresponsive control ELP in Escherichia coli using recombinant DNA techniques. In vivo fluorescence videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors (SKOV-3 ovarian carcinoma and D-54MG glioma) implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately 2-fold increase in tumor localization compared to the same polypeptide without hyperthermia. We observed aggregates of the thermally responsive ELP by fluorescence videomicroscopy within the heated tumor microvasculature but not in control experiments, which demonstrates that the phase transition of the thermally responsive ELP carrier can be engineered to occur in vivo at a specified temperature. By exploiting the phase transition-induced aggregation of these polypeptides, this method provides a new way to thermally target polymer-drug conjugates to solid tumors.
Asunto(s)
Elastina/farmacocinética , Glioma/metabolismo , Hipertermia Inducida , Neoplasias Ováricas/metabolismo , Péptidos/farmacocinética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Estabilidad de Medicamentos , Elastina/química , Elastina/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Microscopía por Video , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Ingeniería de Proteínas , Temperatura , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Patterning of biomolecules on surfaces is an increasingly important technological goal. Because the fabrication of biomolecule arrays often involves stepwise, spatially resolved derivatization of surfaces, spectroscopic imaging of these arrays is important in their fabrication and optimization. Although imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a powerful method for spatially resolved surface analysis, TOF-SIMS images of micropatterned proteins on organic substrates can be difficult to acquire, because of the lack of high intensity, protein-specific molecular ions that are essential for imaging under static conditions. In contrast, low-mass ions are of suitable intensity for imaging, but can originate from different chemical species on the surface. A potential solution to this problem is to utilize stable isotope labeled proteins, an approach that has heretofore not been explored in TOF-SIMS imaging of micropatterned proteins and peptides. To investigate the feasibility of stable isotope enhanced TOF-SIMS imaging of proteins, we synthesized 15N-labeled streptavidin by labeling of the protein during expression from a recombinant gene. The spatial distribution of streptavidin bound to biotin micropatterns, fabricated on a polymer and on a self-assembled monolayer on gold, was imaged by TOF-SIMS. Imaging of high-intensity, low-m/z secondary ions (e.g., C15N-) unique to streptavidin enabled unambiguous spatial mapping of the micropatterned protein with a lateral resolution of a few micrometers. TOF-SIMS imaging of micropatterned 15N-labeled streptavidin also illustrated the exquisite sensitivity of TOF-SIMS to low fractional coverage of protein (5 A effective thickness) in the background regions of the protein micropattern.
Asunto(s)
Estreptavidina/análisis , Procesamiento de Imagen Asistido por Computador , Espectrometría de Masas , Proteínas Recombinantes/químicaRESUMEN
Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.
Asunto(s)
Acrilamidas/química , Materiales Biocompatibles/química , Biopolímeros/química , Ingeniería de Proteínas , Estreptavidina/análogos & derivados , Resinas Acrílicas , Sustitución de Aminoácidos , Distinciones y Premios , Materiales Biocompatibles/efectos de la radiación , Biopolímeros/efectos de la radiación , Fenómenos Químicos , Química Física , Hidrogeles , Concentración de Iones de Hidrógeno , Inmunoensayo/métodos , Luz , Ensayo de Materiales , Estructura Molecular , Mutagénesis Sitio-Dirigida , Sociedades Científicas , Solubilidad , Estreptavidina/química , TemperaturaRESUMEN
There are many protein and DNA based therapeutics under development in the biotechnology and pharmaceutical industries. Key delivery challenges remain before many of these biomolecular therapeutics reach the clinic. Two important barriers are the effective targeting of drugs to specific tissues and cells and the subsequent intracellular delivery to appropriate cellular compartments. In this review, we summarize protein engineering work aimed at improving the stability and refolding efficiency of antibody fragments used in targeting, and at constructing new streptavidin variants which may offer improved performance in pre-targeting delivery strategies. In addition, we review recent work with pH-responsive polymers that mimic the membrane disruptive properties of viruses and toxins. These polymers could serve as alternatives to fusogenic peptides in gene therapy formulations and to enhance the intracellular delivery of protein therapeutics that function in the cytoplasm.
Asunto(s)
Sistemas de Liberación de Medicamentos , Polímeros/química , Ingeniería de Proteínas , Anticuerpos/química , Terapia Genética , Indicadores y Reactivos , Polímeros/síntesis química , Conformación Proteica , Estreptavidina/administración & dosificaciónRESUMEN
Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed "inverse transition cycling," include technical simplicity, low cost, ease of scale-up, and capacity for multiplexing. More broadly, the ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.
Asunto(s)
Péptidos/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Elastina/genética , Elastina/metabolismo , Datos de Secuencia Molecular , Péptidos/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
On the basis of high resolution crystallographic studies of streptavidin and its biotin complex, three principal binding motifs have been identified that contribute to the tight binding. A flexible binding loop can undergo a conformational change from an open to a closed form when biotin is bound. Additional studies described here of unbound wild-type streptavidin have provided structural views of the open conformation. Several tryptophan residues packing around the bound biotin constitute the second binding motif, one dominated by hydrophobic interactions. Mutation of these residues to alanine or phenylalanine have variable effects on the thermodynamics and kinetics of binding, but they generate only small changes in the molecular structure. Hydrogen bonding interactions also contribute significantly to the binding energetics of biotin, and the D128A mutation which breaks a hydrogen bond between the protein and a ureido NH group results in a significant structural alteration that could mimic an intermediate on the dissociation pathway. In this review, we summarize the structural aspects of biotin recognition that have been gained from crystallographic analyses of wild-type and site-directed streptavidin mutants.
Asunto(s)
Estreptavidina/química , Estreptavidina/metabolismo , Sitios de Unión/genética , Biotina/metabolismo , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación Puntual , Conformación Proteica , Ingeniería de Proteínas , Estreptavidina/genéticaRESUMEN
The high affinity energetics in the streptavidin-biotin system provide an excellent model system for studying how proteins balance enthalpic and entropic components to generate an impressive overall free energy for ligand binding. We review here concerted site-directed mutagenesis, biophysical, and computational studies of aromatic and hydrogen bonding interaction energetics between streptavidin and biotin. These results also have provided insight into how streptavidin builds a large activation barrier to dissociation by managing the enthalpic and entropic activation components. Finally, we review recent studies of the biotin dissociation pathway that address the fundamental question of how ligands exit protein binding pockets.
Asunto(s)
Biotina/metabolismo , Estreptavidina/metabolismo , Sitios de Unión , Biotina/química , Biotina/genética , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Estreptavidina/química , Estreptavidina/genética , TermodinámicaRESUMEN
The interaction between streptavidin and its ligand, biotin, were studied by direct force measurements. The complimentary approaches of surface force apparatus (SFA) and atomic force microscopy (AFM) were used to elucidate both long-range and short-range adhesive interactions of the streptavidin biotin interaction. The high spatial resolution of the SFA provided a detailed profile of the intersurface forces of apposing surfaces functionalized with streptavidin and biotin. Measurements obtained by the SFA corresponded to long and intermediate-range forces that are important in determining ligand receptor association. AFM was used to measure the unbinding force of individual streptavidin biotin complexes. These measurements revealed the short-range interactions (i.e. hydrophobic and hydrogen bonding forces) that stabilize the intermolecular bond.
Asunto(s)
Biotina/química , Biotina/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo , Biofisica/instrumentación , Enlace de Hidrógeno , Ligandos , Microscopía de Fuerza Atómica/instrumentación , Unión Proteica , Ingeniería de Proteínas , Propiedades de Superficie , TermodinámicaRESUMEN
Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka approximately 10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.