RESUMEN
Localized drug delivery holds great promise as a means of circumventing traditional chemotherapy side effects associated with high toxicity and prolonged treatments. Nanosized carriers (i.e., with diameters <100 nm) can often accumulate in tumor cells, yet it remains a challenge to design such carriers that are at the same time durable (to survive delivery) and degradable (to release the payload once inside cells). In the present study, photoresponsive catanionic vesicles are utilized to codeliver Bcl-2 siRNA and paclitaxel into MDA-MB-231 human breast cancer cells. These vesicles, which form spontaneously upon simple mixing of an azobenzene-based cationic surfactant and a conventional anionic surfactant, disassociate into free surfactants upon UV illumination. This allows for phototriggered release of the coloaded therapeutics following cellular uptake, which is shown to enhance both cell death and protein suppression. Dynamic light scattering, zeta potential, small-angle neutron scattering, and fluorescence spectroscopy measurements are utilized to determine the optimal vesicle size, charge, bilayer thickness, and concentration for encapsulation and uptake. Cell viability, flow cytometry, and confocal microscopy are used to demonstrate safe and effective dosages, whereas knockdown of Bcl-2 protein expression was confirmed by Western blots.
RESUMEN
We establish and quantify correlations among the molecular structures, interaction forces, and physical processes associated with light-responsive self-assembled surfactant monolayers or bilayers at interfaces. Using the surface forces apparatus (SFA), the interaction forces between adsorbed monolayers and bilayers of an azobenzene-functionalized surfactant can be drastically and controllably altered by light-induced conversion of trans and cis molecular conformations. These reversible conformation changes affect significantly the shape of the molecules, especially in the hydrophobic region, which induces dramatic transformations of molecular packing in self-assembled structures, causing corresponding modulation of electrostatic double layer, steric hydration, and hydrophobic interactions. For bilayers, the isomerization from trans to cis exposes more hydrophobic groups, making the cis bilayers more hydrophobic, which lowers the activation energy barrier for (hemi)fusion. A quantitative and general model is derived for the interaction potential of charged bilayers that includes the electrostatic double-layer force of the Derjaguin-Landau-Verwey-Overbeek theory, attractive hydrophobic interactions, and repulsive steric-hydration forces. The model quantitatively accounts for the elastic strains, deformations, long-range forces, energy maxima, adhesion minima, as well as the instability (when it exists) as two bilayers breakthrough and (hemi)fuse. These results have several important implications, including quantitative and qualitative understanding of the hydrophobic interaction, which is furthermore shown to be a nonadditive interaction.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Tensoactivos/química , Adsorción , Algoritmos , Cinética , Luz , Membrana Dobles de Lípidos/metabolismo , Modelos Químicos , Modelos Moleculares , Conformación Molecular/efectos de la radiación , Compuestos de Amonio Cuaternario/química , Propiedades de Superficie , Tensoactivos/farmacocinéticaRESUMEN
A photoresponsive surfactant has been used as a means to control protein structure and dynamics with light illumination. This cationic azobenzene surfactant, azoTAB, which undergoes a reversible photoisomerization upon exposure to the appropriate wavelength of light, adopts a relatively hydrophobic, trans structure under visible light illumination and a relatively hydrophilic cis structure under UV light illumination. Small-angle neutron scattering (SANS) and neutron spin echo (NSE) spectroscopy were used to measure the tertiary structure and internal dynamics of lysozyme in the presence of the photosurfactant, respectively. The SANS-based in vitro structures indicate that under visible light the photosurfactant induces partial unfolding that principally occurs away from the active site near the hinge region connecting the α and ß domains. Upon UV exposure, however, the protein refolds to a nativelike structure. At the same time, enhanced internal dynamics of lysozyme were detected with the surfactant in the trans form through NSE measurements of the Q-dependent effective diffusion coefficient (D(eff)) of the protein. In contrast, the D(eff) values of lysozyme in the presence of cis azoTAB largely agree with the rigid-body calculation as well as those measured for pure lysozyme, suggesting that the native protein is dormant on the nanosecond time and nanometer length scales. Lysozyme internal motions were modeled by assuming a protein of two (α and ß domains) or three (α and ß domains and the hinge region) domains connects by either soft linkers or rigid, freely rotating bonds. Protein dynamics were also tracked with Fourier transform infrared spectroscopy through hydrogen-deuterium exchange kinetics, which further demonstrated enhanced protein flexibility induced by the trans form of the surfactant relative to the native protein. Ensemble-averaged intramolecular fluorescent resonance energy transfer measurements similarly demonstrated the enhanced dynamics of lysozyme with the trans form of the photosurfactant. Previous results have shown a significant increase in protein activity in the presence of azoTAB in the trans conformation. Combined, these results provide insight into a unique light-based method of controlling protein structure, dynamics, and function and strongly support the relevance of large domain motions for the activity of proteins.
Asunto(s)
Proteínas/química , Animales , Pollos , Medición de Intercambio de Deuterio , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Simulación de Dinámica Molecular , Muramidasa/química , Difracción de Neutrones , Procesos Fotoquímicos , Conformación Proteica , Compuestos de Amonio Cuaternario , Dispersión del Ángulo Pequeño , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Photoreversible changes in the conformation and enzymatic activity of bovine carbonic anhydrase have been investigated as a function of photoresponsive surfactant concentration and light conditions. The light-responsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to photoreversibly control enzyme-surfactant interactions. Small-angle neutron scattering and dynamic light scattering measurements, along with fluorescence spectroscopy, indicate that carbonic anhydrase unfolds upon addition of the surfactant under visible light, while only a small degree of unfolding is observed under UV light. Therefore, the enzyme is completely inactivated in the presence of the trans surfactant, while 40% of the native activity is preserved under UV light, providing a photoreversible "on/off switch" of enzyme activity. Small-angle neutron scattering data provide details of the in vitro conformational changes of the enzyme in response to the photosurfactant and light, with the enzyme found to aggregate as a result of photosurfactant-induced unfolding. Fourier transform infrared (FT-IR) spectroscopy further provides information on the secondary structure changes of the protein in the presence of photosurfactant.
Asunto(s)
Anhidrasas Carbónicas/efectos de la radiación , Tensoactivos/farmacología , Animales , Anhidrasas Carbónicas/efectos de los fármacos , Anhidrasas Carbónicas/metabolismo , Bovinos , Difracción de Neutrones , Conformación Proteica/efectos de la radiación , Pliegue de Proteína/efectos de la radiación , Estructura Secundaria de Proteína/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , Dispersión de Radiación , Dispersión del Ángulo PequeñoRESUMEN
The self-assembly behavior of a light-sensitive azobenzene-based surfactant, both in pure surfactant solutions and in the presence of a hydrophobically modified, water-soluble polymer, has been investigated using small-angle neutron scattering (SANS), light scattering, and UV-vis absorption techniques. The surfactant undergoes reversible photoisomerization upon exposure to the appropriate wavelength of light, with the trans form predominant under visible light being more hydrophobic than the cis isomer under UV-light. As a result, the trans form exhibits a lower critical micelle concentration than does the cis form of the surfactant, allowing photoreversible control of micelle formation. The SANS measurements reveal that micelle formation in pure surfactant solutions with the trans surfactant proceeds as commonly observed in traditional alkyl-based surfactants. Fully developed micelles were observed with aggregation numbers >50, whereas the micelle shapes are consistent with triaxial ellipsoids with axes R(a), R(b), and R(c) approximately equal to 20, 30, and 30-35 A, respectively. In contrast, with the surfactant in the cis conformation disk-shaped premicellar aggregates were observed at low surfactant concentrations with aggregation numbers <10, thicknesses of 6-10 A, and radii of 10-20 A whereas elevated cis-azoTAB concentrations eventually gave rise to fully developed micelles akin to the trans micelles. This stark difference between the self-assembly behavior of the two azobenzene isomers is ascribed to the different geometries of the surfactant in the trans (planar) and cis (bent) conformation. In the presence of the hydrophobically modified polymer, however, both surfactant isomers resulted in well-developed micelles at the respective critical aggregation concentrations (cac's), presumably because of the effect of the dodecyl side chains attached to the polymer on the conformation of the mixed alkyl-azobenzene micelles.
Asunto(s)
Micelas , Difracción de Neutrones , Tensoactivos/química , Compuestos Azo , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Luz , Conformación Molecular/efectos de la radiación , Polímeros , Dispersión del Ángulo Pequeño , Soluciones , Tensoactivos/efectos de la radiación , Rayos UltravioletaRESUMEN
Photoreversible control of the conformation of bacteriorhodopsin in the presence of a light-responsive surfactant is demonstrated through combined UV-vis, FT-IR, and (31)P NMR spectroscopy and dynamic light scattering (DLS) measurements. The azobenzene-based surfactant photoisomerizes upon 434 nm visible (trans, relatively hydrophobic) and 350 nm UV (cis, relatively hydrophilic) illumination, allowing surfactant micellization to be reversibly controlled. This leads to partitioning of the membrane protein into micelles in the unfolded state under visible light, while UV light leads to solubilization of the protein within purple membrane bilayers in the folded state. A three-stage model of purple membrane-photosurfactant interactions is examined through NMR and DLS measurements. Phototriggered unfolding of bacteriorhodopsin, occurring through alpha(II) --> alpha(I) and reverse beta-turn --> extended beta-strand transitions, requires approximately 20 s for completion, while light-induced refolding requires a somewhat longer 80 s as the membrane protein repartitions into the reformed bilayer membrane. Each of these conformational changes can be precisely and reversibly controlled with simple light illumination, providing a novel technique to probe membrane protein folding.
Asunto(s)
Luz , Proteínas de la Membrana/química , Procesos Fotoquímicos , Tensoactivos/farmacología , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Halobacterium salinarum/química , Halobacterium salinarum/metabolismo , Isomerismo , Proteínas de la Membrana/metabolismo , Conformación Proteica/efectos de los fármacos , Conformación Proteica/efectos de la radiación , Pliegue de Proteína/efectos de los fármacos , Pliegue de Proteína/efectos de la radiación , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Tensoactivos/química , Tensoactivos/metabolismoRESUMEN
Photoresponsive catanionic vesicles have been developed as a novel gene delivery vector combining enhanced cellular uptake with phototriggered release of vesicle payload following entry into cells. Vesicles with diameters ranging from 50 to 200 nm [measured using cryo-transmission electron microscopy (TEM) and light-scattering techniques] form spontaneously, following mixing of positively charged azobenzene-containing surfactant and negatively charged alkyl surfactant species. Fluorescent probe measurements showed that the catanionic vesicles at a cation/anion ratio of 7:3 formed at surfactant concentrations as low as 10 microM of the azobenzene surfactant under visible light (with the azobenzene surfactant species principally in the trans configuration), while 50-60 microM of the azobenzene surfactant is required to form vesicles under UV illumination (with the azobenzene surfactant species principally in the cis configuration). At intermediate surfactant concentrations (ca. 15-45 microM) under visible light conditions, transport of DNA-vesicle complexes occurred past the cell membrane of murine fibroblast NIH 3T3 cells through endocytosis. Subsequent UV illumination induced rupture of the vesicles and release of uncomplexed DNA into the cell interiors, where it was capable of passing through the nuclear membrane and thereby contributing to enhanced expression. Single-molecule fluorescent images of T4-DNA demonstrated that the formation of vesicles with a net positive charge led to compaction of DNA molecules via complex formation within a few seconds, while UV-induced disruption of the vesicle-DNA complexes led to DNA re-expansion to the elongated-coil state, also within a few seconds. Transfection experiments with eGFP DNA revealed that photoresponsive catanionic vesicles are more effectively taken up by cells compared to otherwise identical alkyl (i.e., nonazobenzene-containing and thus nonlight-responsive) catanionic vesicles, presumably because of pi-pi stacking interactions that enhance bilayer rigidity in the photoresponsive vesicles. Subsequent UV illumination following endocytosis leads to further dramatic enhancements in the transfection efficiencies, demonstrating that vector unpacking and release of DNA from the carrier complex can be the limiting step in the overall process of gene delivery.
Asunto(s)
Bacteriófago T4/química , ADN Viral/química , Técnicas de Transferencia de Gen , Tensoactivos/química , Rayos Ultravioleta , Animales , Compuestos Azo/química , Cationes , Endocitosis/efectos de la radiación , Humanos , Ratones , Células 3T3 NIHRESUMEN
Direct observation of the mechanism and dynamics of photo-initiated DNA compaction and re-expansion using a light-responsive cationic surfactant has been achieved with fluorescence microscopy. The surfactant undergoes a reversible photoisomerization upon exposure to visible (trans isomer, relatively hydrophobic) or UV (cis isomer, relatively hydrophilic) light. Thus, surfactant binding to DNA and the DNA condensation that result can both be initiated and controlled with light illumination. The inherent kinetics of DNA conformational changes, directly visualized following the in situ light "trigger" of surfactant photoisomerization, are found to occur at rates of approximately 9 microm/s or 240 kbp/s, at or near rates that can be achieved in natural processes. Furthermore, observation of photo-initiated DNA compaction, free of the effects of shear or mixing, provides evidence of a condensation mechanism that nucleates at the ends of the macromolecule. Ethidium bromide displacement studies, employed to gain insight on the mode of interaction between the photo-surfactant and DNA, also reveal the importance of both electrostatic and hydrophobic forces in surfactant binding and DNA condensation.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Compuestos Azo/química , Compuestos Azo/efectos de la radiación , Simulación por Computador , ADN/efectos de la radiación , Cinética , Luz , Microscopía Fluorescente , Estructura Molecular , Fotoquímica , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/efectos de la radiación , Tensoactivos/química , Tensoactivos/efectos de la radiaciónRESUMEN
The effect of an azobenzene-based photoresponsive surfactant on fibril formation of beta-amyloid (1-40) (Abeta40) has been studied using small-angle neutron scattering (SANS), atomic force microscopy (AFM), and light scattering (LS) measurements. Fibril formation is inhibited with a lag phase persisting for approximately 5 h in the presence of the trans isomer of the photosurfactant under visible light (i.e., the relatively hydrophobic, activated form). Conversely, only a 2-h lag phase is observed under UV light with the cis photosurfactant isomer (relatively hydrophilic, passive form), while large fibril networks are immediately observed for the pure protein. Furthermore, in situ UV illumination of a solution of trans surfactant and protein results in rapid fibril formation. Thus, the ability to photoreversibly inhibit and trigger the fibrilization process with light illumination is demonstrated. Shape-reconstruction analysis of the SANS data is used to obtain novel information on the conformation of the protein during the initial stages of protein aggregation. Small, cylindrical protein aggregates 5 nm in diameter and 7 nm long are initially observed during the lag phase independent of the sample conditions. AFM images confirm both the aggregate structure and the duration of the lag phase and further suggest that these early aggregates appear to be the nuclei for longer aggregates that develop over time.
Asunto(s)
Péptidos beta-Amiloides/química , Compuestos Azo/química , Fragmentos de Péptidos/química , Compuestos de Amonio Cuaternario/química , Tensoactivos/química , Luz , Microscopía de Fuerza Atómica , Estructura Molecular , Neutrones , Fotoquímica , Dispersión de Radiación , Factores de TiempoRESUMEN
The interaction of a light-responsive surfactant with lysozyme at pH 5.0 has been investigated as a means to control protein structure and enzymatic activity with light illumination. The cationic azobenzene surfactant undergoes a reversible photoisomerization upon exposure to the appropriate wavelength of light, with the visible-light (trans) form being more hydrophobic and, thus, inducing a greater degree of protein unfolding than the UV-light (cis) form. Conformational changes as a function of photoresponsive surfactant concentration and light illumination were measured through shape-reconstruction analysis of small-angle neutron scattering (SANS) data. The SANS-based in vitro structures indicate that lysozyme transitions from a nativelike structure at low surfactant concentration to a partially unfolded conformation at higher surfactant concentrations under visible light illumination, while UV-light illumination causes the protein to refold to a near-native structure. Protein swelling occurs principally away from the active site near the hinge region connecting the alpha and beta domains, leading to an increase in the observed separation distance of the alpha and beta domains in the ensemble SANS measurements, a likely result of enhanced domain motions and increased flexibility within the protein. This swelling of the hinge region is accompanied by an 8-fold increase in enzymatic activity relative to the native state. Both enzyme swelling and superactivity observed under visible light can be reversed to nativelike conditions upon exposure to UV light, leading to complete photoreversible control of the structure and function of lysozyme.
Asunto(s)
Muramidasa/química , Muramidasa/metabolismo , Animales , Compuestos Azo/química , Quitina/análogos & derivados , Quitina/química , Activación Enzimática/efectos de la radiación , Concentración de Iones de Hidrógeno , Luz , Estructura Molecular , Fotoquímica , Conformación Proteica/efectos de la radiación , Tensoactivos/química , Rayos UltravioletaRESUMEN
Shape-reconstruction analysis applied to small angle neutron scattering (SANS) data is used to determine the in vitro conformations of alpha-chymotrypsin oligomers that form as a result of partial unfolding with a photoresponsive surfactant. In the presence of the photoactive surfactant under visible light, the native oligomers (dimers or compact hexamers) rearrange into expanded corkscrew-like hexamers. Converting the surfactant to the photopassive form with UV light illumination causes the hexamers to laterally aggregate and intertwine into dodecamers with elongated, twisted conformations containing cross-sectional dimensions similar to amyloid protofilaments. Secondary-structure measurements with FT-IR indicate that this photoinduced hexamer-to-dodecamer association occurs through intermolecular beta sheets stabilized with hydrogen bonds, similar to amyloid formation. Traditional structural characterization techniques such as X-ray crystallography and NMR are not easily amenable to the study of these non-native protein conformations; however, SANS is ideally suited to the study of these associated intermediates, providing direct observation of the mechanism of oligomeric formation in an amyloid-forming protein. Combined with photoinitiated hexamer-to-dodecamer associations in the presence of the photoresponsive surfactant, this study could provide unique insight into the amyloidosis disease pathway, as well as novel disease treatment strategies.
Asunto(s)
Amiloide/química , Quimotripsina/química , Compuestos de Amonio Cuaternario/química , Animales , Bovinos , Quimotripsina/efectos de la radiación , Concentración de Iones de Hidrógeno , Modelos Moleculares , Difracción de Neutrones , Dispersión del Ángulo Pequeño , Espectroscopía Infrarroja por Transformada de Fourier , Rayos Ultravioleta , Difracción de Rayos XRESUMEN
The interaction of a light-responsive azobenzene surfactant with bovine serum albumin (BSA) has been investigated as a means to examine photoreversible changes in protein secondary structure. The cationic azobenzene surfactant undergoes a reversible photoisomeriztion upon exposure to the appropriate wavelength of light, with the visible-light (trans) form being more hydrophobic and, thus, inducing a greater degree of protein unfolding than the UV-light (cis) form. Fourier transform infrared (FT-IR) spectroscopy is used to provide quantitative information on the secondary structure elements in the protein (alpha-helices, beta-strands, beta-turns, and unordered domains). Comparing the secondary structure changes induced by light illumination in the presence of the photoresponsive surfactant with previous measurements of the tertiary structure of BSA obtained from small-angle neutron scattering (SANS) allows the three discrete conformation changes in BSA to be fully characterized. At low surfactant concentrations, an alpha-helix --> beta-structure rearrangement is observed as the tertiary structure of BSA changes from a heart-shaped to a distorted heart-shaped conformation. Intermediate surfactant concentrations lead to a dramatic decrease in the alpha-helix fraction in favor of unordered structures, which is accompanied by an unfolding of the C-terminal portion of the protein as evidenced from SANS. Further increases in photosurfactant concentration lead to a beta --> unordered transition with the protein adopting a highly elongated conformation in solution. Each of these protein conformational changes can be precisely and reversibly controlled with light illumination, as revealed through FT-IR spectra collected during repeated visible-light <--> UV-light cycles.
Asunto(s)
Compuestos Azo/química , Luz , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/efectos de la radiación , Tensoactivos/química , Animales , Bovinos , Estructura Molecular , Fotoquímica , Estructura Secundaria de Proteína , Sensibilidad y Especificidad , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
A means to control DNA compaction with light illumination has been developed using the interaction of DNA with a photoresponsive cationic surfactant. The surfactant undergoes a reversible photoisomerization upon exposure to visible (trans isomer, more hydrophobic) or UV (cis isomer, more hydrophilic) light. As a result, surfactant binding to DNA and the resulting DNA condensation can be tuned with light. Dynamic light scattering (DLS) measurements were used to follow lambda-DNA compaction from the elongated-coil to the compact globular form as a function of surfactant addition and light illumination. The results reveal that compaction occurs at a surfactant-to-DNA base pair ratio of approximately 7 under visible light, while no compaction is observed up to a ratio of 31 under UV light. Upon compaction, the measured diffusion coefficient increases from a value of 0.6 x 10(-8) cm2/s (elongated coil with an end-to-end distance of 1.27 microm) to a value of 1.7 x 10(-8) cm2/s (compact globule with a hydrodynamic radius of 120 nm). Moreover, the light-scattering results demonstrate that the compaction process is completely photoreversible. Fluorescence microscopy with T4-DNA was used to further confirm the light-scattering results, allowing single-molecule detection of the light-controlled coil-to-globule transition. These structural studies were combined with absorbance and fluorescence spectroscopy of crystal violet in order to elucidate the binding mechanism of the photosurfactant to DNA. The results indicate that both electrostatic and hydrophobic forces are important in the compaction process. Finally, a DNA-photosurfactant-water phase diagram was constructed to examine the effects of both DNA and surfactant concentration on DNA compaction. The results reveal that precipitation, which occurs during the latter stages of condensation, can also be reversibly controlled with light illumination. The combined results clearly show the ability to control the interaction between DNA and the complexing agent and, therefore, DNA condensation with light.
Asunto(s)
ADN/metabolismo , Fotoquímica , Tensoactivos/efectos de la radiación , Precipitación Química , Interacciones Hidrofóbicas e Hidrofílicas , Luz , Conformación de Ácido Nucleico , Estereoisomerismo , Tensoactivos/metabolismoRESUMEN
A means to control lysozyme conformation with light illumination has been developed using the interaction of the protein with a photoresponsive surfactant. Upon exposure to the appropriate wavelength of light, the azobenzene surfactant undergoes a reversible photoisomerization, with the visible-light (trans) form being more hydrophobic than the UV-light (cis) form. As a result, surfactant binding to the protein and, thus, protein unfolding, can be tuned with light. Small-angle neutron scattering (SANS) measurements were used to provide detailed information of the protein conformation in solution. Shape-reconstruction methods applied to the SANS data indicate that under visible light the protein exhibits a native-like form at low surfactant concentrations, a partially swollen form at intermediate concentrations, and a swollen/unfolded form at higher surfactant concentrations. Furthermore, the SANS data combined with FT-IR spectroscopic analysis of the protein secondary structure reveal that unfolding occurs primarily in the alpha domain of lysozyme, while the beta domain remains relatively intact. Thus, the surfactant-unfolded intermediate of lysozyme appears to be a separate structure than the well-known alpha-domain intermediate of lysozyme that contains a folded alpha domain and unfolded beta domain. Because the interactions between the photosurfactant and protein can be tuned with light, illumination with UV light returns the protein to a native-like conformation. Fluorescence emission data of the nonpolar probe Nile red indicate that hydrophobic domains become available for probe partitioning in surfactant-protein solutions under visible light, while the availability of these hydrophobic domains to the probe decrease under UV light. Dynamic light scattering and UV-vis spectroscopic measurements further confirm the shape-reconstruction findings and reveal three discrete conformations of lysozyme. The results clearly demonstrate that visible light causes a greater degree of lysozyme swelling than UV light, thus allowing for the protein conformation to be controlled with light.
Asunto(s)
Muramidasa/química , Muramidasa/efectos de la radiación , Conformación Proteica , Pliegue de Proteína , Compuestos Azo/química , Compuestos Azo/efectos de la radiación , Luz , Oxazinas/química , Oxazinas/efectos de la radiación , Fotoquímica , Fármacos Fotosensibilizantes/farmacología , Conformación Proteica/efectos de la radiación , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/efectos de la radiación , Dispersión de Radiación , Espectrometría de Fluorescencia , Espectrofotometría , Espectroscopía Infrarroja por Transformada de Fourier , Tensoactivos/química , Tensoactivos/efectos de la radiaciónRESUMEN
The photoresponsive interaction of light-sensitive azobenzene surfactants with bovine serum albumin (BSA) at neutral pH has been investigated as a means to control protein folding with light irradiation. The cationic azobenzene surfactant undergoes a reversible photoisomerization upon exposure to the appropriate wavelength of light, with the visible-light (trans) form of the surfactant being more hydrophobic than the UV-light (cis) form. As a consequence, the trans form exhibits enhanced interaction with the protein compared to the cis form of the surfactant, allowing photoreversible control of the protein folding/unfolding phenomena. Small-angle neutron-scattering (SANS) measurements are used to provide detailed information on the protein conformation in solution. A fitting of the protein shape to a low-resolution triaxial ellipsoid model indicates that three discrete forms of the protein exist in solution depending on the surfactant concentration, with lengths of approximately 90, 150, and 250 A, respectively, consistent with additional dynamic light-scattering measurements. In addition, shape-reconstruction methods are applied to the SANS data to obtain relatively high-resolution conformation information. The results confirm that BSA adopts a heart-shaped structure in solution at low surfactant concentration, similar to the well-known X-ray crystallographic structure. At intermediate surfactant concentrations, protein elongation results as a consequence of the C-terminal portion separating from the rest of the molecule. Further increases in the surfactant concentration eventually lead to a highly elongated protein that nonetheless still exhibits some degree of folding that is consistent with the literature observations of a relatively high helical content in denatured BSA. The results clearly demonstrate that the visible-light form of the surfactant causes a greater degree of protein unfolding than the UV-light form, providing a means to control protein folding with light that, within the resolution of SANS, appears to be completely reversible.
Asunto(s)
Luz , Fármacos Fotosensibilizantes/química , Pliegue de Proteína , Albúmina Sérica Bovina/química , Tensoactivos/química , Compuestos Azo/química , Compuestos Azo/metabolismo , Compuestos Azo/efectos de la radiación , Colorantes Fluorescentes/química , Modelos Químicos , Neutrones , Oxazinas/química , Fotoquímica , Fármacos Fotosensibilizantes/metabolismo , Fármacos Fotosensibilizantes/efectos de la radiación , Conformación Proteica , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/metabolismo , Compuestos de Amonio Cuaternario/efectos de la radiación , Dispersión de Radiación , Albúmina Sérica Bovina/metabolismo , Albúmina Sérica Bovina/efectos de la radiación , Tensoactivos/metabolismo , Tensoactivos/efectos de la radiación , Rayos UltravioletaRESUMEN
Liquid and supercritical carbon dioxide swell potassium carboxylate perfluoropolyether (PFPE-K) cylindrical micelles in water to produce novel CO(2)-in-water (C/W) microemulsions. The swelling elongates the micelles significantly from 20 to 80 nm as the molar ratio of CO(2) in the micelles to surfactant (R(CO2)) reaches approximately 8. As the micelles swell to form microemulsions, the solubility of pyrene increases by a factor of ca. 10. Fluorescence spectra suggest that pyrene resides primarily in the low-polarity micelle core rather than in the palisade region. The results illustrate the ability of C/W microemulsions to solubilize both lipophilic and fluorophilic substances simultaneously.