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BACKGROUND: Each year approximately five million people die from injuries. In countries where systems of trauma care have been introduced, death and disability have decreased. A major component of developed trauma systems is a trauma quality improvement (TQI) program and trauma quality improvement meeting (TQIM). Effective TQIMs improve trauma care by identifying and fixing problems. But globally, TQIMs are absent or unstructured in most hospitals providing trauma care. The aim of this study was to implement and evaluate a checklist for a structured TQIM. METHODS: This project was conducted as a prospective before-and-after study in four major trauma centres in India. The intervention was the introduction of a structured TQIM using a checklist, introduced with a workshop. This workshop was based on the World Health Organization (WHO) TQI Programs short course and resources, plus the developed TQIM checklist. Pre- and post-intervention data collection occurred at all meetings in which cases of trauma death were discussed. The primary outcome was TQIM Checklist compliance, defined by the discussion of, and agreement upon each of the following: preventability of death, identification of opportunities to improve care and corrective actions and a plan for closing the loop. RESULTS: There were 34 meetings in each phase, with 99 cases brought to the pre-intervention phase and 125 cases brought to the post-intervention phase. There was an increase in the proportion of cases brought to the meeting for which preventability of death was discussed (from 94% to 100%, p = 0.007) and agreed (from 7 to 19%, OR 3.7; 95% CI:1.4-9.4, p = 0.004) and for which a plan for closing the loop was discussed (from 2% to 18%, OR 10.9; 95% CI:2.5-47.6, p < 0.001) and agreed (from 2% to 18%, OR 10.9; 95% CI:2.5-47.6, p < 0.001). CONCLUSION: This study developed, implemented and evaluated a TQIM Checklist for improving TQIM processes. The introduction of a TQIM Checklist, with training, into four Indian trauma centres, led to more structured TQIMs, including increased discussion and agreement on preventability of death and plans for loop closure. A TQIM Checklist should be considered for all centres managing trauma patients.
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Adhesión a Directriz , Mejoramiento de la Calidad/normas , Centros Traumatológicos , Heridas y Lesiones/terapia , Lista de Verificación , Congresos como Asunto , Medicina Basada en la Evidencia , Humanos , India/epidemiología , Guías de Práctica Clínica como Asunto , Estudios Prospectivos , Heridas y Lesiones/epidemiologíaRESUMEN
PURPOSE: Abdominal wall hernias are a common problem. The success of abdominal wall reconstruction decreases with increasing hernia size. This study summarizes the outcomes of one surgeon's experience using a "sandwich" technique for hernia repair in patients with loss of abdominal domain. METHODS: We reviewed our ventral hernia repair (VHR) experience from 2008 to 2015 among patients with loss of domain, as defined by a hernia defect greater than 300 cm2. The percent of herniation through the defect, defined by a hernia sac-to-abdominal cavity volume ratio, was measured on preoperative CT scans by four independent reviewers and averaged. Outcomes were compared among those with giant ventral hernias (hernia sac-to-abdominal cavity volume >30%) and those with smaller defect ratios. RESULTS: Over the study period, 21 patients underwent VHR. In 17 patients (81%), a "sandwich" technique was utilized. Ten patients had hernia sac-to-abdominal cavity defects less than 30%, and 11 had defects greater than 30%. Preoperative characteristics were similar in both groups with the exception of a higher ASA score in those with giant ventral hernias and more Ventral Hernia Working Group Grade 3 hernias in those without giant ventral hernias. Postoperative outcomes were similar in both groups. There were no mortalities. There were two recurrences (18%) in the giant VHR group and none in the smaller defect group (p = 0.16). Surgical site occurrences were noted in 48% of patients and did not differ between giant and non-giant VHR groups (50 vs 45%, p = 0.84). Average postoperative length of stay was significantly longer in the giant VHR group (31 vs. 17 days, p = 0.03). CONCLUSIONS: Our results suggest that the "sandwich" technique for VHR is a safe and durable method to restore abdominal wall integrity in those with LOD, even in patients with giant ventral hernias.
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Pared Abdominal/cirugía , Hernia Ventral/cirugía , Herniorrafia/métodos , Cavidad Abdominal/diagnóstico por imagen , Cavidad Abdominal/patología , Pared Abdominal/diagnóstico por imagen , Pared Abdominal/patología , Dermis Acelular , Femenino , Hernia Ventral/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Tamaño de los Órganos , Estudios Retrospectivos , Mallas QuirúrgicasRESUMEN
BACKGROUND: Ventral hernia repair (VHR) is a commonly performed operation, but analysis of patient outcomes based upon hernia size is lacking. We sought to identify differences in operative repair and post-operative morbidity and mortality after open VHR based on hernia defect size. METHODS: Patient and operative data were retrospectively reviewed on all patients undergoing open incisional VHR between January 2008 and February 2015 by a single surgeon at the Johns Hopkins Hospital. Patient variables were described by means for continuous variables and percentages for discrete variables, with differences between groups calculated by Chi-squared analysis. RESULTS: During the study period, 228 patients underwent open VHR during which intraoperative defect size was measured. Patients were split into four groups based upon defect size: less than 200 cm2, 200-300 cm2, 301-400 cm2, and over 400 cm2. Patients with large defects were more likely to present with a recurrent hernia (P = 0.007) and trended towards a history of wound infections (P = 0.07). Operative time was significantly longer as defect size increased (P < 0.001). Component separation was most frequently used in patients with defects 200-300 cm2 in size (P = 0.001), in whom primary closure was most likely to occur. While mesh was used in almost all patients, the specific location (overlay only, underlay only, or overlay with underlay) depended on hernia size (P < 0.001). Mean length of stay increased with defect size (P < 0.001). Larger defect size was associated with increased 30-day morbidity (P = 0.03) but not readmission (P = 0.53), recurrence (P = 0.99), or mortality (P = 0.99). CONCLUSION: Hernia defect size affects operative time and surgical technique for repair of a ventral hernia. Larger defect size is associated with increased post-operative morbidity and length of stay but not readmission, recurrence, or mortality. Hernia size greater than 400 cm2 should not be a limitation to operative repair.
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Pared Abdominal/patología , Hernia Ventral/patología , Hernia Ventral/cirugía , Herniorrafia/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Pesos y Medidas Corporales , Femenino , Herniorrafia/métodos , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Mallas Quirúrgicas , Adulto JovenRESUMEN
AIMS: To examine the potential of magnetic nanoparticles (MNPs) in transfecting human osteosarcoma fibroblasts (MG-63) and investigate the effects of a novel non-viral oscillating nanomagnetic gene transfection system (magnefect-nano™) in enhancing transfection efficiency (TE). METHODS: MG-63 cells were transfected using MNPs coupled with a GFP-carrying plasmid. The magnefect-nano system was evaluated for transfection efficiency and potential associated effects on cell viability. RESULTS: MG-63 cells were efficiently transfected using MNPs and the magnefect-nano system significantly enhanced overall transfection efficiency. MNPs were not found to affect cell viability and/or function of the cells. CONCLUSION: Non-viral transfection using MNPs and the magnefect-nano system can be used to transfect MG-63 cells and assist reporter gene delivery on a single cell basis, highlighting the wide potential of nanomagnetic gene transfection in gene therapy.
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Campos Magnéticos , Nanopartículas/química , Osteoblastos/citología , Transfección/métodos , Línea Celular Tumoral , Membrana Celular/metabolismo , Supervivencia Celular , ADN/química , Electroporación , Endosomas/química , Citometría de Flujo , Terapia Genética , Proteínas Fluorescentes Verdes , Humanos , Lípidos/química , Magnetismo , Microscopía Fluorescente , Nanotecnología/métodos , OscilometríaRESUMEN
Genetic therapies for cystic fibrosis (CF) must be assessed for safety and efficacy, so testing in a non-human primate (NHP) model is invaluable. In this pilot study we determined if the conducting airways of marmosets (n = 2) could be transduced using an airway pre-treatment followed by an intratracheal bolus dose of a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector (LacZ reporter). LacZ gene expression (X-gal) was assessed after 7 days and found primarily in conducting airway epithelia as well as in alveolar regions. The LacZ gene was not detected in liver or spleen via qPCR. Vector p24 protein bio-distribution into blood was transient. Dosing was well tolerated. This preliminary study confirmed the transducibility of CF-relevant airway cell types. The marmoset is a promising NHP model for testing and translating genetic treatments for CF airway disease towards clinical trials.
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Callithrix/virología , Técnicas de Transferencia de Gen , Operón Lac/genética , Lentivirus/genética , Transducción Genética/métodos , Animales , Fibrosis Quística/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Femenino , Expresión Génica , Vectores Genéticos , Pulmón/metabolismo , Pulmón/virología , Masculino , Proyectos PilotoRESUMEN
UNLABELLED: The objective of this work was to examine the effects of magnet distance (and by proxy, field strength) on nanomagnetic transfection efficiency. METHODS: non-viral magnetic nanoparticle-based transfection was evaluated using both static and oscillating magnet arrays. RESULTS: Fluorescence intensity (firefly luciferase) of transfected H292 cells showed no increase using a 96-well NdFeB magnet array when the magnets were 5 mm from the cell culture plate or nearer. At 6 mm and higher, fluorescence intensity decreased systematically. CONCLUSION: In all cases, fluorescence intensity was higher when using an oscillating array compared to a static array. For distances closer than 5 mm, the oscillating system also outperformed Lipofectamine 2000™.
RESUMEN
Attempts have been made to use various forms of cellular vectors to deliver therapeutic genes to diseased tissues like malignant tumours. However, this approach has proved problematic due to the poor uptake of these vectors by the target tissue. We have devised a novel way of using magnetic nanoparticles (MNPs) to enhance the uptake of such 'therapeutically armed' cells by tumours. Monocytes naturally migrate from the bloodstream into tumours, so attempts have been made to use them to deliver therapeutic genes to these sites. However, transfected monocytes injected systemically fail to infiltrate tumours in large numbers. Using a new in vitro assay for assessing monocyte extravasation, we show that the ability of transfected human monocytes to migrate across a human endothelial cell layer into a 3D tumour spheroid is markedly increased when cells are pre-loaded with MNPs and a magnetic force is applied close to the spheroid. Furthermore, systemic administration of such 'magnetic' monocytes to mice bearing solid tumours led to a marked increase in their extravasation into the tumour in the presence of an external magnet. This new magnetic targeting approach could be used to increase the targeting, and thus the efficacy, of many cell-based gene therapies in vivo.
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Terapia Genética/métodos , Magnetismo , Monocitos/metabolismo , Nanopartículas , Neoplasias/terapia , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Células Cultivadas , Células Endoteliales/fisiología , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hierro , Masculino , Ratones , Ratones Desnudos , Microscopía Fluorescente , Neoplasias/patología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Fagocitosis , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.
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Morfolinas/química , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Antígenos CD/química , Antígenos CD/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , Fotoquímica , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.
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Fragmentos de Péptidos/genética , Péptidos/genética , Transfección/métodos , Secuencia de Aminoácidos , Animales , Técnicas de Cultivo de Célula , ADN/genética , Células Dendríticas/metabolismo , Endotelio Vascular/citología , Vectores Genéticos , Humanos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Péptidos/química , Células Tumorales CultivadasRESUMEN
Uptake of polycation-DNA particles is the first step in achieving gene delivery with non-viral vehicles. One of the important characteristics determining uptake of DNA particles is their size. Here we have characterized the ability of several cell lines to internalise labelled polystyrene microspheres of different sizes. All the cell lines tested ingested 20-nm microspheres avidly. With larger microspheres (93, 220, 560 and 1010 nm) cell type as well as growth related differences were observed. Whereas some cell lines (HUVEC, ECV 304 and HNX 14C) took up microspheres up to 1010 nm even when the cells were confluent, others did not take up many microspheres larger than 93 nm (Hepa 1-6 and HepG2). In one cell line (KLN 205), uptake of 93-, 220- and 560-nm microspheres was avid in growing cells, but not detectable when they were confluent. In KLN 205 cells, a good correlation was found between the uptake of 560-nm microspheres and the uptake of a peptide-DNA polyplex formulation, when it was prepared under conditions leading to small particle sizes. Little correlation was found when the polyplex formulation was allowed to aggregate.
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Poliestirenos , Secuencia de Aminoácidos , Animales , Línea Celular , Colagenasas , ADN/química , Matriz Extracelular , Humanos , Hidrólisis , Ratones , Microesferas , Datos de Secuencia Molecular , Oligopéptidos/administración & dosificación , Oligopéptidos/metabolismo , Tamaño de la PartículaRESUMEN
Nuclear MR spectroscopy in low and medium magnetic fields yields well-resolved natural abundance proton and decoupled phosphorus spectra from small (1-10 mL) volumes of brain in vivo in minutes. With this tool, neurochemical research has advanced through identification and noninvasve assay of a specific neuronal-cf2Ncf1-acetylaspartate, glial (cf2myocf1-inositol)-markers, energetics and osmolutes, and neurotransmitters (glutamate, GABA). From these simple measurements, several dozen disease states are recognized, including birth injury, and white matter and Alzheimer disease. Together, these tools are having a major impact on neuroscience and clinical medicine.
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Enfermedad de Alzheimer/diagnóstico , Espectroscopía de Resonancia Magnética , Química Encefálica , Humanos , Inositol/análisis , Espectroscopía de Resonancia Magnética/métodosRESUMEN
Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a ubiquitin-protein ligase containing multiple WW domains. We have previously demonstrated the association between the WW domains of Nedd4 and PPxY (PY) motifs of the epithelial sodium channel (ENaC). In this paper, we report the assignment of backbone 1H alpha, 1HN, 15N, 13C', 13C alpha, and aliphatic 13C resonances of a fragment of rat Nedd4 (rNedd4) containing the two C-terminal WW domains, WW(II+III), complexed to a PY motif-containing peptide derived from the beta subunit of rat ENaC, the betaP2 peptide. The secondary structures of these two WW domains, determined from chemical shifts of 13C alpha and 13C beta resonances, are virtually identical to those of the WW domains of the Yes-associated protein YAP65 and the peptidyl-prolyl isomerase Pin1. Triple resonance experiments that detect the 1H alpha chemical shift were necessary to complete the chemical shift assignment, owing to the large number of proline residues in this fragment of rNedd4. A new experiment, which correlates sequential residues via their 15N nuclei and also detects 1H alpha chemical shifts, is introduced and its utility for the chemical shift assignment of sequential proline residues is discussed. Data collected on the WW(II+III)-betaP2 complex indicate that these WW domains have different affinities for the betaP2 peptide.
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Proteínas de Unión al Calcio/química , Ligasas , Espectroscopía de Resonancia Magnética , Estructura Secundaria de Proteína , Canales de Sodio/química , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Canales Epiteliales de Sodio , Datos de Secuencia Molecular , Ubiquitina-Proteína Ligasas Nedd4 , Fragmentos de Péptidos/metabolismo , Prolina/química , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The backbone dynamics of both folded and unfolded states of staphylococcal nuclease (SNase) and the N-terminal SH3 domain from drk (drkN SH3) are studied at two different temperatures. A simple method for obtaining order parameters, describing the amplitudes of motion of bond vectors, from NMR relaxation measurements of both folded and unfolded proteins is presented and the data obtained for 15N-NH bond vectors in both the SNase and drkN SH3 systems analyzed with this approach. Using a recently developed theory relating the amplitude of bond vector motions to conformational entropy, the entropy change between the folded and unfolded forms of SNase is calculated on a per residue basis. It is noteworthy that the region of the molecule with the smallest entropy change includes those residues showing native-like structure in the unfolded form of the molecule, as established by NOE-based experiments. Order parameters of backbone 15N-NH bond vectors show significantly larger changes with temperature in the unfolded states of both proteins relative to the corresponding folded forms. The differential temperature dependence is interpreted in terms of differences in the heat capacities of folded and unfolded polypeptide chains. The contribution to the heat capacity of the unfolded chain from rapid 15N-NH bond vector motions is calculated and compared with estimates of the heat capacity of the backbone unit, -CHCONH-, obtained from calorimetric data. Methyl dynamics measured at 14 and 30 degrees C establish that the amplitudes of side-chain motions in the folded SH3 domain are more sensitive to changes in temperature than the backbone dynamics, suggesting that over this temperature range side-chain ps to ns time-scale motions contribute more to the heat capacity than backbone motions for this protein.
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Proteínas de Drosophila , Proteínas de Insectos/química , Nucleasa Microcócica/química , Conformación Proteica , Dominios Homologos src , Entropía , Espectroscopía de Resonancia Magnética , Matemática , Pliegue de Proteína , Temperatura , TermodinámicaRESUMEN
Pulse sequences are presented for the measurement of 3JC'C gamma and 3JNC gamma scalar couplings for all C gamma containing residues in 15N,13C uniformly labeled proteins. The methods described are based on quantitative J correlation spectroscopy pioneered by Bax and co-workers [Bax et. al. (1994) Methods Enzymol., 239, 79-105]. The combination of 3JC'C gamma and 3JNC gamma scalar coupling constants allows the assignment of discrete rotameric states about the chi 1 torsion angle in cases where such states exist or, alternatively, facilitates the establishment of noncanonical chi 1 conformations or the presence of rotameric averaging. The methods are applied to a 1.5 mM sample of staphylococcal nuclease.
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Isótopos de Carbono , Espectroscopía de Resonancia Magnética/métodos , Nucleasa Microcócica/química , Modelos Químicos , Isótopos de Nitrógeno , Aminoácidos/química , Conformación ProteicaRESUMEN
Measurements of 15N NMR relaxation parameters have been used to characterize the backbone dynamics of folded and denatured states of the N-terminal SH3 domain from the adapter protein drk, in high salt or guanidinium chloride, respectively. Values of the spectral density function evaluated at a number of frequencies are compared. The levels of backbone dynamics in the folded protein show little variation across the molecule and are of similar magnitude to those determined previously for the folded state of the protein in exchange with an unfolded state at low salt concentrations [Farrow et al. (1995) Biochemistry 34, 868-878]. The denatured state of the domain exhibits both more extensive and more heterogeneous dynamics than the folded state. In particular the profile of the spectral density function evaluated at zero-frequency for the unfolded state of the domain indicates that residues in the middle of the protein sequence are considerably less mobile than those at the termini. These data suggest that the molecule is not behaving as an extended polymer and that concerted motions of the central portions of the molecule are occurring, consistent with a reasonably compact conformation in this region. The backbone dynamics of the folded and unfolded states were studied at two temperatures. The level of high-frequency motions in the folded molecule is largely unaffected by changes in temperature, whereas an increase in temperature results in increased high-frequency motion in the unfolded state.
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Proteínas de Drosophila , Pliegue de Proteína , Estructura Terciaria de Proteína , Dominios Homologos src , Animales , Tampones (Química) , Drosophila , Guanidina , Guanidinas , Hormonas de Insectos/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Desnaturalización Proteica , SulfatosRESUMEN
Nuclear magnetic resonance spectroscopy (MRS) in low and medium magnetic fields yields well-resolved natural abundance proton and decoupled phosphorus spectra from small (1-10 cc) volumes of brain in vivo in minutes. With this tool, neurochemical research has advanced through identification and non-invasive assay of specific neuronal--(N-acetylaspartate), glial (myo-inositol)--markers, energetics and osmolytes, and neurotransmitters (glutamate, GABA). From these simple measurements, several dozen disease states are recognized, including birth injury, and white matter and Alzheimer disease. Addition of stable isotopes of carbon (in man) or nitrogen (in experimental animals) has provided in vivo assays of enzyme flux through glucose transport, glycolysis, TCA-cycle, and the glutamine-glutamate-GABA system. Finally, a number of xenobiotics are recognized with heteronuclear NMR techniques. Together, these tools are having a major impact on neuroscience and clinical medicine. Through diagnosis and therapeutic monitoring, a new generation of in vivo metabolite imaging is expected with the advent of conforming RF coils and higher field NMR systems.
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Encéfalo/metabolismo , Demencia/diagnóstico , Demencia/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Animales , Fenómenos Biofísicos , Biofisica , HumanosRESUMEN
The structure and dynamics of the 53-residue filamentous bacteriophage IKe major coat protein in fully protonated myristoyllysophosphatidylglycerol (MPG) micelles were characterized using multinuclear solution NMR spectroscopy. Detergent-solubilized coat protein [sequence: see text] mimics the membrane-bound "assembly intermediate" form of the coat protein which occurs during part of the phage life cycle. NMR studies of the IKe coat protein show that the coat protein is largely alpha-helical, exhibiting a long amphipathic surface. helix (Asn 4 to Ser 26) and a shorter "micelle-spanning" C-terminal helix which begins at TRP 29 and continues at least to Phe 48. Pro 30 likely occurs in the first turn of the C-terminal helix, where it is ideally situated given the hydrogen bonding and steric restrictions imposed by this residue. The similarity of 15N relaxation values (T1, T2, and NOE and 500 MHz and T2 at 600 MHz) among much of the N-terminal helix and all of the TM helix indicates that the N-terminal helix is as closely associated with the micelle as the TM helix. The description of the protein in the micelle is supported by the observation of NOEs between lysolipid protons and protein amide protons between asn 8 and Ser 50. The N-terminal and TM helices exhibit substantial mobility on the microsecond to second time scale, which likely reflects changes in the orientation between the two helices. The overall findings serve to clarify the role of individual residues in the context of a TM alpha-helix and provide an understanding of the secondary structure, dynamics, and aqueous and micellar environments of the coat protein.
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Proteínas de la Cápside , Cápside/química , Inovirus/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Lisofosfolípidos/química , Espectroscopía de Resonancia Magnética , Micelas , Modelos Moleculares , Datos de Secuencia Molecular , SolucionesRESUMEN
Protein-protein interfaces can consist of interactions between large numbers of residues of each molecule; some of these interactions are critical in determining binding affinity and conferring specificity, while others appear to play only a marginal role. Src-homology-2 (SH2) domains bind to proteins containing phosphorylated tyrosines, with additional specificity provided by interactions with residues C-terminal to the phosphotyrosine (pTyr) residue. While the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2) interacts with eight residues of a pTyr-containing peptide from its high affinity binding site on the beta-platelet-derived growth factor receptor, it can still bind tightly to a phosphopeptide containing only three residues. Novel deuterium (2H) based nuclear magnetic resonance (NMR) spin relaxation experiments which probe the nanosecond-picosecond time scale dynamics of methyl containing side chain residues have established that certain regions of the PLCC SH2 domain contacting the residues C-terminal to the pTyr have a high degree of mobility in both the free and peptide complexed states. In contrast, there is significant restriction of motion in the pTyr binding site. These results suggest a correlation between the dynamic behavior of certain groups in the PLCC SH2 complex and their contribution to high affinity binding and binding specificity.
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Dominios Homologos src/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Técnicas In Vitro , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfolipasa C delta , Unión Proteica , Conformación Proteica , Receptores del Factor de Crecimiento Derivado de Plaquetas/química , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Termodinámica , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo , Dominios Homologos src/genéticaRESUMEN
A method is presented for the determination of values of the spectral density function, J(omega), describing the dynamics of amide bond vectors from 15N relaxation parameters alone. Assuming that the spectral density is given by the sum of Lorentzian functions, the approach allows values of J(omega) to be obtained at omega = 0, omega N and 0.870 omega H, where omega N and omega H are Larmor frequencies of nitrogen and proton nuclei, respectively, from measurements of 15N T1, T2 and 1H-15N steady-state NOE values at a single spectrometer frequency. Alternatively, when measurements are performed at two different spectrometer frequencies of i and j MHz, J(omega) can be mapped at omega = 0, omega iN, omega jN, 0.870 omega iH and 0.870 omega iH, where omega iN, for example, is the 15N Larmor frequency for a spectrometer operating at 1 MHz. Additionally, measurements made at two different spectrometer frequencies enable contributions to transverse relaxation from motions on millisecond-microsecond time scales to be evaluated and permit assessment of whether a description of the internal dynamics is consistent with a correlation function consisting of a sum of exponentials. No assumptions about the specific form of the spectral density function describing the dynamics of the 15N-NH bond vector are necessary, provided that dJ(omega)/d omega is relatively constant between omega = omega H + omega N to omega = omega H - omega N. Simulations demonstrate that the method is accurate for a wide range of protein motions and correlation times, and experimental data establish the validity of the methodology. Results are presented for a folded and an unfolded form of the N-terminal SH3 domain of the protein drk.