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1.
Nat Chem Biol ; 12(2): 94-101, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26656088

RESUMEN

Protein aggregation underlies an array of human diseases, yet only one small-molecule therapeutic targeting this process has been successfully developed to date. Here, we introduce an in vivo system, based on a ß-lactamase tripartite fusion construct, that is capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid ß) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split ß-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of islet amyloid polypeptide aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation.


Asunto(s)
Bioensayo/métodos , Agregación Patológica de Proteínas , Péptidos beta-Amiloides/metabolismo , Western Blotting , Curcumina/farmacología , Dopamina/química , Dopamina/farmacología , Humanos , Microscopía Electrónica de Transmisión , Unión Proteica/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray , beta-Lactamasas/química
2.
Langmuir ; 26(2): 1002-7, 2010 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-19785459

RESUMEN

In single molecule force measurements with soft atomic force microscope (AFM) cantilevers, the force sensitivity is limited by the Brownian motion of the cantilever. When a cantilever is close to the surface, the hydrodynamic interaction between the cantilever beam and the surface, called the "squeezing effect", becomes significant, and the resonance peak of the thermal oscillation of the cantilever is heavily broadened and shifted to lower frequency which makes it difficult to eliminate the thermal noise by low-pass filtering. In this study, we propose an easy and low-cost method to improve the force sensitivity. We demonstrate that by bringing a tip of a cantilever onto the edge of a micropillar structure a significant reduction of the damping and an enhancement of force sensitivity are achieved.


Asunto(s)
Microscopía de Fuerza Atómica/instrumentación , Diseño de Equipo/instrumentación , Modelos Teóricos , Termodinámica
3.
Biophys J ; 96(6): 2415-27, 2009 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-19289066

RESUMEN

Removal of Bbetal-42 from fibrinogen by Crotalus atrox venom results in a molecule lacking fibrinopeptide B and part of a thrombin binding site. We investigated the mechanism of polymerization of desBbeta1-42 fibrin. Fibrinogen trinodular structure was clearly observed using high resolution noncontact atomic force microscopy. E-regions were smaller in desBbeta1-42 than normal fibrinogen (1.2 nm +/- 0.3 vs. 1.5 nm +/- 0.2), whereas there were no differences between the D-regions (1.7 nm +/- 0.4 vs. 1.7 nm +/- 0.3). Polymerization rate for desBbeta1-42 was slower than normal, resulting in clots with thinner fibers. Differences in oligomers were found, with predominantly lateral associations for desBbeta1-42 and longitudinal associations for normal fibrin. Clot elasticity as measured by magnetic tweezers showed a G' of approximately 1 Pa for desBbeta1-42 compared with approximately 8 Pa for normal fibrin. Spring constants of early stage desBbeta1-42 single fibers determined by atomic force microscopy were approximately 3 times less than normal fibers of comparable dimensions and development. We conclude that Bbeta1-42 plays an important role in fibrin oligomer formation. Absence of Bbeta1-42 influences oligomer structure, affects the structure and properties of the final clot, and markedly reduces stiffness of the whole clot as well as individual fibrin fibers.


Asunto(s)
Fibrinógeno/química , Fibrinógeno/metabolismo , Sitios de Unión , Elasticidad , Electroforesis en Gel de Poliacrilamida , Humanos , Microscopía de Fuerza Atómica , Multimerización de Proteína , Análisis Espectral , Trombina/metabolismo
4.
Biophys J ; 94(4): 1326-40, 2008 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-17933878

RESUMEN

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l(d)) and raft liquid ordered (l(o)) lipid phases by selectively solubilizing the l(d) phase. A higher concentration of Lubrol was required, and not all the l(d) phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l(d) phase and then progressed to the solubilization of both l(d) and l(o) phases simultaneously. Octyl glucoside simultaneously solubilized both l(o) and l(d) phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.


Asunto(s)
Membrana Celular/química , Detergentes/química , Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Modelos Químicos , Fosfolípidos/química , Animales , Células CHO , Cricetinae , Cricetulus , Solubilidad
5.
Appl Spectrosc ; 62(10): 1060-9, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18926013

RESUMEN

By using near-infrared surface-enhanced Raman scattering (SERS) with 60 nm gold nanoparticles (Au-NPs) to probe the chemical composition inside single human osteosarcoma cells we have shown that the SERS intensity may increase by a factor of 3-6 times in different parts of the cells depending on the density of gold nanoaggregates within the probed volume after the cell is dehydrated. The cellular points of low-density gold nanoaggregates exhibit more significant increase of SERS signal levels, the cellular macrochemicals such as nucleic acids show conformational changes, and new components can be probed after the cell is completely dried. A comparative study between viable and apoptotic cells indicates that most of the Au-NPs that enter the living cell reside in the cytoplasm and around the nucleus, whereas glyoxal-induced apoptotic cells show relatively uniform distribution of Au-NPs and, interestingly, the presence of DNA fragments is detected throughout the cell, including the cell surface.


Asunto(s)
Biomarcadores de Tumor/análisis , Oro , Nanopartículas/ultraestructura , Osteosarcoma/química , Espectrometría Raman/métodos , Línea Celular Tumoral , Humanos , Tamaño de la Partícula , Propiedades de Superficie
6.
Nucleic Acids Res ; 34(9): 2698-709, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16714447

RESUMEN

DNA packaging in the bacteriophage phi29 involves a molecular motor with protein and RNA components, including interactions between the viral connector protein and molecules of pRNA, both of which form multimeric complexes. Data are presented to demonstrate the higher order assembly of pRNA together with the affinity of pRNA:pRNA and pRNA:connector interactions, which are used to propose a model for motor function. In solution, pRNA can form dimeric and trimeric multimers in a magnesium-dependent manner, with dissociation constants for multimerization in the micromolar range. pRNA:connector binding is also facilitated by the presence of magnesium ions, with a nanomolar apparent dissociation constant for the interaction. From studies with a mutant pRNA, it appears that multimerization of pRNA is not essential for connector binding and it is likely that connector protein is involved in the stabilization of higher order RNA multimers. It is proposed that magnesium ions may promote conformational change that facilitate pRNA:connector interactions, essential for motor function.


Asunto(s)
Fagos de Bacillus/genética , Proteínas de la Cápside/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Ensamble de Virus , Fagos de Bacillus/fisiología , Secuencia de Bases , ADN Viral/química , Datos de Secuencia Molecular
7.
J Mol Biol ; 351(4): 850-64, 2005 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-16024039

RESUMEN

Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of beta2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.


Asunto(s)
Amiloide/química , Microglobulina beta-2/química , Amiloide/metabolismo , Amiloide/ultraestructura , Humanos , Técnicas In Vitro , Cinética , Microscopía de Fuerza Atómica , Modelos Moleculares , Complejos Multiproteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Microglobulina beta-2/metabolismo , Microglobulina beta-2/ultraestructura
8.
J Mol Biol ; 330(4): 785-97, 2003 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-12850147

RESUMEN

The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.


Asunto(s)
Amiloide/química , Microglobulina beta-2/química , División Celular , Humanos , Concentración de Iones de Hidrógeno , Cinética , Microscopía de Fuerza Atómica , Unión Proteica , Proteínas Recombinantes/química , Factores de Tiempo
9.
Protein Sci ; 11(12): 2759-65, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12441375

RESUMEN

The mechanical resistance of a folded domain in a polyprotein of five mutant I27 domains (C47S, C63S I27)(5)is shown to depend on the unfolding history of the protein. This observation can be understood on the basis of competition between two effects, that of the changing number of domains attempting to unfold, and the progressive increase in the compliance of the polyprotein as domains unfold. We present Monte Carlo simulations that show the effect and experimental data that verify these observations. The results are confirmed using an analytical model based on transition state theory. The model and simulations also predict that the mechanical resistance of a domain depends on the stiffness of the surrounding scaffold that holds the domain in vivo, and on the length of the unfolded domain. Together, these additional factors that influence the mechanical resistance of proteins have important consequences for our understanding of natural proteins that have evolved to withstand force.


Asunto(s)
Pliegue de Proteína , Proteínas/química , Simulación por Computador , Modelos Moleculares , Método de Montecarlo , Estructura Terciaria de Proteína , Proteínas/metabolismo
11.
J Phys Chem B ; 114(30): 9912-9, 2010 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-20666531

RESUMEN

Thermal folding/unfolding kinetics of wild-type ubiquitin (wt-UBQ) was studied in a wide time range, from microseconds to seconds, by combining rapid-mixing T-jump and laser T-jump with fluorescence detection (MTJ-F and LTJ-F, respectively) to monitor the fluorescence changes of Tyr-59 located on the 310-helix. The kinetics occurs exclusively in the millisecond to second time range, and the decays are strictly single exponential. From global analysis of folding and unfolding decays, the kf and ku values were determined, without use of the equilibrium constant Ku. The activation enthalpy of folding is negative (DeltaHf(#)(Tm) = -10.8 kcal/mol), but the free energy of the transition state is substantially larger than that of the unfolded state (DeltaGf(#)(Tm) = 7.6 kcal/mol RTm). Thus, wt-UBQ behaves as a two-state folder, when folding is monitored by the fluorescence of Tyr-59. The observation of kinetics on the microsecond time scale, when folding is monitored by the disruption of hydrogen bonds between beta-strands, using nonlinear infrared spectroscopy of the amide I vibrations (LTJ-DVE) [Chung, H. S.; Tokmakoff, A. Proteins: Struct., Funct., Bioinf. 2008, 72, 474-487], seems to result from the fact that MTJ-F monitors the effective unfolding (backbone exposure to water) of the thermally excited protein alone, while LTJ-DVE also monitors preliminary events (hydrogen-bond breaking) and thermal re-equilibration of the thermally excited protein.


Asunto(s)
Tirosina/química , Ubiquitina/química , Animales , Rastreo Diferencial de Calorimetría , Bovinos , Fluorescencia , Cinética , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Temperatura , Termodinámica , Factores de Tiempo
12.
J Mol Biol ; 398(1): 132-45, 2010 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-20211187

RESUMEN

Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I(eqm)) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I(eqm) variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na(2)SO(4) in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding.


Asunto(s)
Proteínas Portadoras/química , Proteínas de Escherichia coli/química , Pliegue de Proteína , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Simulación por Computador , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Cinética , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Urea/farmacología
13.
Nat Struct Mol Biol ; 16(3): 318-24, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19252485

RESUMEN

Many proteins reach their native state through pathways involving the presence of folding intermediates. It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints. Here, using protein-engineering, ultra-rapid mixing and stopped-flow experiments combined with restrained molecular dynamics simulations, we characterize the transition state for the formation of the intermediate populated during the folding of the bacterial immunity protein, Im7, and the subsequent molecular steps leading to the native state. The results provide a comprehensive view of the folding process of this small protein. An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.


Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Pliegue de Proteína , Cinética , Modelos Moleculares , Estructura Terciaria de Proteína
14.
Head Neck Oncol ; 1: 38, 2009 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-19863815

RESUMEN

Raman spectroscopy could offer non-invasive, rapid and an objective nature to cancer diagnostics. However, much work in this field has focused on resolving differences between cancerous and non-cancerous tissues, and lacks the reproducibility and interpretation to be put into clinical practice. Much work is needed on basic cellular differences between malignancy and normal. This would allow the establishment of a clinically relevant cellular based model to translate to tissue classification. Raman spectroscopy provides a very detailed biochemical analysis of the target material and to 'unlock' this potential requires sophisticated mathematical modelling such as neural networks as an adjunct to data interpretation. Commercially obtained cancerous and non-cancerous cells, cultured in the laboratory were used in Raman spectral measurements. Data trends were visualised through PCA and then subjected to neural network analysis based on self-organising maps; consisting of m maps, where m is the number of classes to be recognised. Each map approximates the statistical distribution of a given class. The neural network analysis provided a 95% accuracy for identification of the cancerous cell line and 92% accuracy for normal cell line. In this preliminary study we have demonstrated th ability to distinguish between "normal" and cancerous commercial cell lines. This encourages future work to establish the reasons underpinning these spectral differences and to move forward to more complex systems involving tissues. We have also shown that the use of sophisticated mathematical modelling allows a high degree of discrimination of 'raw' spectral data.


Asunto(s)
Redes Neurales de la Computación , Espectrometría Raman/métodos , Glándula Tiroides/citología , Neoplasias de la Tiroides/patología , Línea Celular , Humanos , Modelos Teóricos , Análisis de Componente Principal
15.
Head Neck Oncol ; 1: 34, 2009 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-19761601

RESUMEN

Cancer poses a massive health burden with incidence rates expected to double globally over the next decade. In the United Kingdom screening programmes exists for cervical, breast, and colorectal cancer. The ability to screen individuals for solid malignant tumours using only a peripheral blood sample would revolutionise cancer services and permit early diagnosis and intervention. Raman spectroscopy interrogates native biochemistry through the interaction of light with matter, producing a high definition biochemical 'fingerprint' of the target material. This paper explores the possibility of using Raman spectroscopy to discriminate between cancer and non-cancer patients through a peripheral blood sample. Forty blood samples were obtained from patients with Head and Neck cancer and patients with respiratory illnesses to act as a positive control. Raman spectroscopy was carried out on all samples with the resulting spectra being used to build a classifier in order to distinguish between the cancer and respiratory patients' spectra; firstly using principal component analysis (PCA)/linear discriminant analysis (LDA), and secondly with a genetic evolutionary algorithm. The PCA/LDA classifier gave a 65% sensitivity and specificity for discrimination between the cancer and respiratory groups. A sensitivity score of 75% with a specificity of 75% was achieved with a 'trained' evolutionary algorithm. In conclusion this preliminary study has demonstrated the feasibility of using Raman spectroscopy in cancer screening and diagnostics of solid tumours through a peripheral blood sample. Further work needs to be carried out for this technique to be implemented in the clinical setting.


Asunto(s)
Detección Precoz del Cáncer/métodos , Neoplasias/diagnóstico , Espectrometría Raman/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Análisis de Componente Principal
16.
J Mol Biol ; 384(1): 264-78, 2008 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-18805425

RESUMEN

We have examined the folding ensembles present in solution for a series of RNA oligonucleotides that encompass the replicase translational operator stem-loop of the RNA bacteriophage MS2. Single-molecule (SM) fluorescence assays suggest that these RNAs exist in solution as ensembles of differentially base-paired/base-stacked states at equilibrium. There are two distinct ensembles for the wild-type sequence, implying the existence of a significant free energy barrier between "folded" and "unfolded" ensembles. Experiments with sequence variants are consistent with an unfolding mechanism in which interruptions to base-paired duplexes, in this example by the single-stranded loop and a single-base bulge in the base-paired stem, as well as the free ends, act as nucleation points for unfolding. The switch between folded and unfolded ensembles is consistent with a transition that occurs when all base-pairing and/or base-stacking interactions that would orientate the legs of the RNA stem are broken. Strikingly, a U-to-C replacement of a residue in the loop, which creates a high-affinity form of the operator for coat protein binding, results in dramatically different (un)folding behaviour, revealing distinct subpopulations that are either stabilised or destabilised with respect to the wild-type sequence. This result suggests additional reasons for selection against the C-variant stem-loop in vivo and provides an explanation for the increased affinity.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Levivirus/química , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/metabolismo , Secuencia de Bases , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Cinética , Datos de Secuencia Molecular , Mutación/genética , Estructura Secundaria de Proteína , ARN Viral/genética , Análisis Espectral , Termodinámica
17.
Faraday Discuss ; 139: 35-51; discussion 105-28, 419-20, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19048989

RESUMEN

Experiments that measure the viscoelasticity of single molecules from the Brownian fluctuations of an atomic force microscope (AFM) have provided a new window onto their internal dynamics in an underlying conformational landscape. Here we develop and apply these methods to examine the internal friction of unfolded polypeptide chains at high stretch. The results reveal a power law dependence of internal friction with tension (exponent 1.3 +/- 0.5) and a relaxation time approximately independent of force. To explain these results we develop a frictional worm-like chain (FWLC) model based on the Rayleigh dissipation function of a stiff chain with dynamical resistance to local bending. We analyse the dissipation rate integrated over the chain length by its Fourier components to calculate an effective tension-dependent friction constant for the end-to-end vector of the chain. The result is an internal friction that increases as a power law with tension with an exponent 3/2, consistent with experiment. Extracting the intrinsic bending friction constant of the chain it is found to be approximately 7 orders of magnitude greater than expected from solvent friction alone; a possible explanation we offer is that the underlying energy landscape for bending amino acids and/or peptide bond is rough, consistent with recent results on both proteins and polysaccharides.


Asunto(s)
Péptidos/química , Elasticidad , Fricción , Viscosidad
18.
Blood ; 111(2): 643-50, 2008 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-17925485

RESUMEN

Fibrinogen BbetaArg448Lys is a common polymorphism, positioned within the carboxyl terminus of the Bbeta-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. This study demonstrates for the first time that clots made from recombinant BbetaLys448 fibrinogen are characterized by thin fibers and small pores, show increased stiffness, and appear more resistant to fibrinolysis. Fibrinogen BbetaArg448Lys is a primary example of common genetic variation with a significant phenotypic effect at the molecular level.


Asunto(s)
Sustitución de Aminoácidos , Fibrina/química , Fibrinógeno/química , Modelos Moleculares , Polimorfismo Genético , Animales , Células COS , Chlorocebus aethiops , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Fibrina/genética , Fibrina/metabolismo , Fibrina/ultraestructura , Fibrinógeno/genética , Fibrinógeno/metabolismo , Fibrinólisis/genética , Humanos , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología
19.
Mol Membr Biol ; 24(3): 233-42, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17520480

RESUMEN

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.


Asunto(s)
Colesterol/química , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Microdominios de Membrana/ultraestructura , Esfingomielinas/química , Animales , Células CHO , Colesterol/metabolismo , Cricetinae , Cricetulus , Detergentes/química , Dipeptidasas/química , Dipeptidasas/metabolismo , Riñón/química , Riñón/metabolismo , Membrana Dobles de Lípidos/metabolismo , Liposomas , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Microscopía de Fuerza Atómica/métodos , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Fosfatidilcolinas , Solubilidad , Esfingomielinas/metabolismo , Porcinos
20.
Anal Chem ; 79(10): 3646-53, 2007 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-17441678

RESUMEN

We report on the capabilities of near-infrared surface-enhanced Raman scattering (SERS) using gold nanoparticles to obtain detailed chemical information with high spatial resolution from within single cancer cells, living or fixed. Colloidal gold particles, 60 nm in size, were introduced into live human osteosarcoma cells by endocytosis by adding them to the growth medium. Rapid SERS mapping of cells indicated that not only could rich vibrational spectra be obtained from intrinsic cellular constituents both in the cytoplasm and nucleus and but also the distribution of extrinsic molecules introduced into the cells, in this case, rhodamine 6G could be characterized, suggesting that the intracellular distribution of chemotherapeutic agents could potentially be measured by this technique. We show that the SERS signal intensity from the cellular components increases and more spectral detail is acquired from dried cells when compared with hydrated cells in buffer. The data also show spectral fluctuations, mainly in intensity but also in peak position, which are dependent upon the intensity of the excitation light and are probably due to diffusion of molecules on the surface of the gold nanoparticles. A detailed understanding of the origins of these effects is still not complete, but the ability to acquire very sensitive SERS inside living cancer cells indicates the potential of this technique as a useful tool in biomedicine.


Asunto(s)
Oro/farmacocinética , Nanopartículas del Metal , Sondas Moleculares , Osteosarcoma/patología , Espectrometría Raman/métodos , Línea Celular Tumoral , Núcleo Celular , Citoplasma , Humanos , Osteosarcoma/ultraestructura , Rodaminas/farmacocinética
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