RESUMEN
BACKGROUND: Levels of stress post-injury, especially after compensable injury, are known to be associated with worse long-term recovery. It is therefore important to identify how, and in whom, worry and stress manifest post-injury. This study aimed to identify demographic, injury, and compensation factors associated with worry about financial and recovery outcomes 12 months after traumatic injury. METHODS: Participants (nâ¯=â¯433) were recruited from the Victorian Orthopaedic Trauma Outcomes Registry and Victorian State Trauma Registry after admission to a major trauma hospital in Melbourne, Australia. Participants completed questionnaires about pain, compensation experience and psychological wellbeing as part of a registry-based observational study. RESULTS: Linear regressions showed that demographic and injury factors accounted for 11% and 13% of variance in financial and recovery worry, respectively. Specifically, lower education, discharge to inpatient rehabilitation, attributing fault to another and having a compensation claim predicted financial worry. Worry about recovery was only predicted by longer hospital stay and attributing fault to another. In all participants, financial and recovery worry were associated with worse pain (severity, interference, catastrophizing, kinesiophobia, self-efficacy), physical (disability, functioning) and psychological (anxiety, depression, PTSD, perceived injustice) outcomes 12 months post-injury. In participants who had transport (nâ¯=â¯135) or work (nâ¯=â¯22) injury compensation claims, both financial and recovery worry were associated with sustaining permanent impairments, and reporting negative compensation system experience 12 months post-injury. Financial worry 12 months post-injury was associated with not returning to work by 3-6 months post-injury, whereas recovery worry was associated with attributing fault to another, and higher healthcare use at 6-12 months post-injury. CONCLUSIONS: These findings highlight the important contribution of factors other than injury severity, to worry about finances and recovery post-injury. Having a compensation claim, failure to return to work and experiencing pain and psychological symptoms also contribute to elevated worry. As these factors explained less than half of the variance in worry, however, other factors not measured in this study must play a role. As worry may increase the risk of developing secondary mental health conditions, timely access to financial, rehabilitation and psychological supports should be provided to people who are not coping after injury.
Asunto(s)
Personas con Discapacidad/rehabilitación , Reinserción al Trabajo/psicología , Heridas y Lesiones/rehabilitación , Adulto , Anciano , Ansiedad , Compensación y Reparación , Evaluación de la Discapacidad , Personas con Discapacidad/psicología , Femenino , Financiación Personal , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Sistema de Registros , Reinserción al Trabajo/economía , Reinserción al Trabajo/estadística & datos numéricos , Apoyo Social , Victoria/epidemiología , Heridas y Lesiones/economía , Heridas y Lesiones/epidemiología , Heridas y Lesiones/psicología , Adulto JovenRESUMEN
Purpose The objective of this systematic review was to synthesize evidence on the effectiveness of workplace-based return-to-work (RTW) interventions and work disability management (DM) interventions that assist workers with musculoskeletal (MSK) and pain-related conditions and mental health (MH) conditions with RTW. Methods We followed a systematic review process developed by the Institute for Work & Health and an adapted best evidence synthesis that ranked evidence as strong, moderate, limited, or insufficient. Results Seven electronic databases were searched from January 1990 until April 2015, yielding 8898 non-duplicate references. Evidence from 36 medium and high quality studies were synthesized on 12 different intervention categories across three broad domains: health-focused, service coordination, and work modification interventions. There was strong evidence that duration away from work from both MSK or pain-related conditions and MH conditions were significantly reduced by multi-domain interventions encompassing at least two of the three domains. There was moderate evidence that these multi-domain interventions had a positive impact on cost outcomes. There was strong evidence that cognitive behavioural therapy interventions that do not also include workplace modifications or service coordination components are not effective in helping workers with MH conditions in RTW. Evidence for the effectiveness of other single-domain interventions was mixed, with some studies reporting positive effects and others reporting no effects on lost time and work functioning. Conclusions While there is substantial research literature focused on RTW, there are only a small number of quality workplace-based RTW intervention studies that involve workers with MSK or pain-related conditions and MH conditions. We recommend implementing multi-domain interventions (i.e. with healthcare provision, service coordination, and work accommodation components) to help reduce lost time for MSK or pain-related conditions and MH conditions. Practitioners should also consider implementing these programs to help improve work functioning and reduce costs associated with work disability.
Asunto(s)
Trastornos Mentales/rehabilitación , Dolor Musculoesquelético/rehabilitación , Reinserción al Trabajo , Absentismo , Terapia Cognitivo-Conductual/métodos , Estudios de Cohortes , Humanos , Enfermedades Profesionales/economía , Enfermedades Profesionales/rehabilitación , Traumatismos Ocupacionales/economía , Traumatismos Ocupacionales/rehabilitación , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Work on the perception of urban soundscapes has generated a number of perceptual models which are proposed as tools to test and evaluate soundscape interventions. However, despite the excessive sound levels and noise within hospital environments, perceptual models have not been developed for these spaces. To address this, a two-stage approach was developed by the authors to create such a model. First, semantics were obtained from listening evaluations which captured the feelings of individuals from hearing hospital sounds. Then, 30 participants rated a range of sound clips representative of a ward soundscape based on these semantics. Principal component analysis extracted a two-dimensional space representing an emotional-cognitive response. The framework enables soundscape interventions to be tested which may improve the perception of these hospital environments.
Asunto(s)
Estimulación Acústica/psicología , Percepción Auditiva , Emociones , Ambiente de Instituciones de Salud , Hospitales , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Teóricos , Ruido , Análisis de Componente Principal , Semántica , Adulto JovenRESUMEN
BACKGROUND: Pain is a common presenting complaint and there is considerable debate regarding the best practice for analgesia in the pre-hospital environment for trauma patients with severe pain. METHODS: A review of the literature was conducted using a number of electronic medical literature databases from their earliest record to the latest available at the time the search was conducted (May 2010). Medical Subject Headings, keywords and a pre-hospital search filter were used to yield relevant literature. RESULTS: The search strategy yielded a total of 837 references. Seven hundred and fifty of these references were excluded as they did not meet the inclusion criteria. Of the 87 articles short listed for abstract or full-text review, six reported on ketamine use as an analgesic agent in the pre-hospital setting. Two papers were prospective randomized-controlled trials, and the number of patients included in the studies ranged from 4 to 164. Three studies aimed to report on the effectiveness of ketamine for pain intensity reduction; two concluded that ketamine provided safe and effective pain relief and one reported that ketamine reduced the amount of morphine required but was not associated with a reduction in pain intensity. One study identified a significantly higher prevalence of adverse effects following ketamine administration. The other studies reported no significant side effects and concluded that ketamine was safe. CONCLUSION: Ketamine is a safe and effective analgesic agent. The addition of ketamine as an analgesic agent may improve the management of patients presenting with acute traumatic pain in the pre-hospital setting.
Asunto(s)
Analgésicos/uso terapéutico , Servicios Médicos de Urgencia , Ketamina/uso terapéutico , Dolor/tratamiento farmacológico , Heridas y Lesiones/fisiopatología , Analgésicos/efectos adversos , Humanos , Ketamina/efectos adversos , Ensayos Clínicos Controlados Aleatorios como Asunto , Heridas y Lesiones/tratamiento farmacológicoRESUMEN
BACKGROUND: The identification and monitoring of critical incidents or adverse events and error reporting is a relatively new area of study in the prehospital setting. In 2005, we commenced a prospective descriptive study of the implementation of a Critical Incident Monitoring process in a rural/regional pre-hospital setting. The objective of the project was to describe the nature and incidence of errors detected in the management of prehospital trauma with the ultimate aim of identifying processes to reduce or mitigate such incidents. This paper describes the barriers to reporting critical incidents identified during the 3-year study. METHOD: This study used a qualitative approach involving the triangulation of a number of ethnographic methodologies, including unscripted focus groups, informal interviews and qualitative aspects of surveys utilised in a broader research project. Prevailing themes were fed back to participants in an iterative process to further explore perceptions and beliefs regarding these concepts. The final analysis of themes is descriptively presented. RESULTS: A number of barriers were identified and categorised into seven themes. These themes were; Burden of reporting, fear of disciplinary action, fear of potential litigation, fear of breaches of confidentiality and fear of embarrassment, concern that 'nothing would change' even if the incident was reported, lack of familiarity with process and impact of 'blame culture'. CONCLUSION: There are numerous barriers to reporting critical incidents. One of the key approaches which may alleviate many of the barriers to reporting is shifting to a systems based focus rather than an individual 'shame and blame' approach. The underlying barriers lie in the culture of the profession, and appear consistent across other health care disciplines.
Asunto(s)
Barreras de Comunicación , Servicios Médicos de Urgencia/métodos , Notificación Obligatoria , Errores Médicos/estadística & datos numéricos , Gestión de Riesgos/organización & administración , Estudios Transversales , Femenino , Encuestas de Atención de la Salud , Humanos , Masculino , Errores Médicos/prevención & control , Evaluación de Necesidades , VictoriaRESUMEN
Severe pain is a common presenting symptom for emergency patients. One major challenge in the management of severe pain is the objective measurement of pain. Due to the subjective nature of pain, it can be very difficult for clinicians to quantify pain intensity and measure the qualitative features of the pain experience. A number of measurement tools have been validated in the acute care setting, with some appropriate for use in the prehospital setting. This paper reviews the characteristics required of a prehospital acute pain measure and appraises the relative utility of a number of currently used pain measures. At present, the verbal numerical rating scale appears the most appropriate pain measure to administer in the prehospital setting for adult patients as it is practical and valid. Either the Oucher scale or the faces pain scale is suitable for prehospital care providers to assess pain in children.
Asunto(s)
Servicios Médicos de Urgencia , Dimensión del Dolor/métodos , Dolor/diagnóstico , Enfermedad Aguda , Adulto , Presión Sanguínea/fisiología , Frecuencia Cardíaca/fisiología , Humanos , Fenómenos Fisiológicos Respiratorios , Encuestas y Cuestionarios , VictoriaRESUMEN
Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pK(a) approximately 7. An alpha-helix is formed at high pH spanning 8 residues of a 21-residue-long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, called the activation loop-helix, which leads to the formation and stability of the alpha-helix, pK(a) values of all ionizable residues of GART have been calculated, using Poisson-Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pK(a) values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pK(a) calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helix-promoting sequence Arg122-Gln123-Ala124-Leu125-Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic-site residue His108, to facilitate catalysis.
Asunto(s)
Transferasas de Hidroximetilo y Formilo/química , Alanina/química , Aminoácidos/química , Catálisis , Cristalografía por Rayos X , Estabilidad de Enzimas , Histidina/química , Concentración de Iones de Hidrógeno , Transferasas de Hidroximetilo y Formilo/genética , Transferasas de Hidroximetilo y Formilo/fisiología , Matemática , Modelos Moleculares , Mutación , Fosforribosilglicinamida-Formiltransferasa , Conformación Proteica , Electricidad Estática , VolumetríaRESUMEN
The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (fTHF) to glycinamide ribonucleotide (GAR), a process that is pH-dependent with pK(a) of approximately 8. Experimental studies of pH-rate profiles of wild-type and site-directed mutants of GART have led to the proposal that His108, Asp144, and GAR are involved in catalysis, with His108 being an acid catalyst, while forming a salt bridge with Asp144, and GAR being a nucleophile to attack the formyl group of fTHF. This model implied a protonated histidine with pK(a) of 9.7 and a neutral GAR with pK(a) of 6.8. These proposed unusual pK(a)s have led us to investigate the electrostatic environment of the active site of GART. We have used Poisson-Boltzmann-based electrostatic methods to calculate the pK(a)s of all ionizable groups, using the crystallographic structure of a ternary complex of GART involving the pseudosubstrate 5-deaza-5,6,7,8-THF (5dTHF) and substrate GAR. Theoretical mutation and deletion analogs have been constructed to elucidate pairwise electrostatic interactions between key ionizable sites within the catalytic site. Also, a construct of a more realistic catalytic site including a reconstructed pseudocofactor with an attached formyl group, in an environment with optimal local van der Waals interactions (locally minimized) that imitates closely the catalytic reactants, has been used for pK(a) calculations. Strong electrostatic coupling among catalytic residues His108, Asp144, and substrate GAR was observed, which is extremely sensitive to the initial protonation and imidazole ring flip state of His108 and small structural changes. We show that a proton can be exchanged between GAR and His108, depending on their relative geometry and their distance to Asp144, and when the proton is attached on His108, catalysis could be possible. Using the formylated locally minimized construct of GART, a high pK(a) for His108 was calculated, indicating a protonated histidine, and a low pK(a) for GAR(NH(2)) was calculated, indicating that GAR is in neutral form. Our results are in qualitative agreement with the current mechanistic picture of the catalytic process of GART deduced from the experimental data, but they do not reproduce the absolute magnitude of the pK(a)s extracted from fits of k(cat)-pH profiles, possibly because the static time-averaged crystallographic structure does not describe adequately the dynamic nature of the catalytic site during binding and catalysis. In addition, a strong effect on the pK(a) of GAR(NH(2)) is produced by the theoretical mutations of His108Ala and Asp144Ala, which is not in agreement with the observed insensitivity of the pK(a) of GAR(NH(2)) modeled from the experimental data using similar mutations. Finally, we show that important three-way electrostatic interactions between highly conserved His137, with His108 and Asp144, are responsible for stabilizing the electrostatic microenvironment of the catalytic site. In conclusion, our data suggest that further detailed computational and experimental work is necessary.
Asunto(s)
Glicina/análogos & derivados , Transferasas de Hidroximetilo y Formilo/química , Bombas de Protones/química , Sitios de Unión , Catálisis , Cristalografía , Glicina/química , Histidina/química , Concentración de Iones de Hidrógeno , Transferasas de Hidroximetilo y Formilo/genética , Matemática , Fosforribosilglicinamida-Formiltransferasa , Protones , Ribonucleótidos/química , Electricidad Estática , VolumetríaRESUMEN
The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. The clamp proteins also participate in a variety of dynamic and competing protein-protein interactions. However, clamp-protein binding sequences have not so far been identified in the eubacteria. Here we show from three lines of evidence, bioinformatics, yeast two-hybrid analysis, and inhibition of protein-protein interaction by modified peptides, that variants of a pentapeptide motif (consensus QL[SD]LF) are sufficient to enable interaction of a number of proteins with an archetypal eubacterial sliding clamp (the beta subunit of Escherichia coli DNA polymerase III holoenzyme). Representatives of this motif are present in most sequenced members of the eubacterial DnaE, PolC, PolB, DinB, and UmuC families of DNA polymerases and the MutS1 mismatch repair protein family. The component tripeptide DLF inhibits the binding of the alpha (DnaE) subunit of E. coli DNA polymerase III to beta at microM concentration, identifying key residues. Comparison of the eubacterial, eukaryotic, and archaeal sliding clamp binding motifs suggests that the basic interactions have been conserved across the evolutionary landscape.
Asunto(s)
Proteínas Bacterianas/genética , Reparación del ADN , Replicación del ADN , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cromosomas Bacterianos/genética , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Bases de Datos Factuales , Desoxirribonucleasa EcoRI/metabolismo , Herpesvirus Humano 1/genética , Cinética , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Biosíntesis de Proteínas , Subunidades de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Recent theoretical and experimental studies have suggested that real proteins have sequences with sufficiently small energetic frustration that topological effects are central in determining the folding mechanism. A particularly interesting and challenging framework for exploring and testing the viability of these energetically unfrustrated models is the study of circular-permuted proteins. Here we present the results of the application of a topology-based model to the study of circular permuted SH3 and CI2, in comparison with the available experimental results. The folding mechanism of the permuted proteins emerging from our simulations is in very good agreement with the experimental observations. The differences between the folding mechanisms of the permuted and wild-type proteins seem then to be strongly related to the change in the native state topology.
Asunto(s)
Simulación por Computador , Ingeniería de Proteínas , Pliegue de Proteína , Proteínas/química , Proteínas/metabolismo , Sitios de Unión , Modelos Moleculares , Mutación , Péptidos/química , Péptidos/metabolismo , Proteínas de Plantas , Probabilidad , Termodinámica , Dominios Homologos src , Familia-src Quinasas/química , Familia-src Quinasas/metabolismoRESUMEN
The specificity of intracellular signaling events is controlled, in part, by compartmentalization of protein kinases and phosphatases. The subcellular localization of these enzymes is often maintained by protein- protein interactions. A prototypic example is the compartmentalization of the cAMP-dependent protein kinase (PKA) through its association with A-kinase anchoring proteins (AKAPs). A docking and dimerization domain (D/D) located within the first 45 residues of each regulatory (R) subunit protomer forms a high affinity binding site for its anchoring partner. We now report the structures of two D/D-AKAP peptide complexes obtained by solution NMR methods, one with Ht31(493-515) and the other with AKAP79(392-413). We present the first direct structural data demonstrating the helical nature of the peptides. The structures reveal conserved hydrophobic interaction surfaces on the helical AKAP peptides and the PKA R subunit, which are responsible for mediating the high affinity association in the complexes. In a departure from the dimer-dimer interactions seen in other X-type four-helix bundle dimeric proteins, our structures reveal a novel hydrophobic groove that accommodates one AKAP per RIIalpha D/D.
Asunto(s)
Proteínas Portadoras/química , Proteínas Quinasas Dependientes de AMP Cíclico/química , Péptidos/química , Secuencia de Aminoácidos , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Péptidos y Proteínas de Señalización Intercelular , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , SolucionesRESUMEN
Interleukin-1 beta (IL-1 beta) is a cytokine and a member of the beta-trefoil superfamily of protein structures. An interesting feature in the folding of IL-1 beta, shared with some other members of the same topological family, is the existence of a slow step in folding to the native conformation from a discrete intermediate. Wanting to probe the nature of this slow step in the folding of WT IL-1 beta (tau(1)=45 seconds), we made ten sequence variants of IL-1 beta (L10A, T9Q, T9G, C8S, C8A, N7G, N7D, L6A, R4P, and R4Q), where all mutations are located along strand 1. This strand is not protected from hydrogen exchange until late in folding. Most of the mutations showed little effect on the kinetics of folding for IL-1 beta. However, C8 is clearly involved in both the late and the early steps in folding, while sequence variants at L10 and L6 affect only late events in folding. The value of the slowest relaxation time, tau(1), which is associated with the rate of native protein formation, increased for the refolding of C8S, while C8A, L6A, and L10A showed smaller but systematic increases in the value of tau(1.)For both C8S and C8A, the value of the step associated with formation of the intermediate, tau(2), was independent of denaturant concentration. In addition, mutations in the hydrophobic core (L10A, C8A, C8S, and L6A) and, surprisingly, along the surface (T9G, T9Q, and N7G) alter the stability. The most destabilizing mutations show changes in equilibrium unfolding cooperativity, which is atypical for destabilizing mutations in IL-1 beta. Crystallographic studies indicate that mutations along strand 1 may alter the number of ordered water molecules within the core. Thus, side-chain replacement in this region can disrupt essential main-chain interactions mediated by ordered water contacts in a highly cooperative network of hydrogen bonding.
Asunto(s)
Interleucina-1/química , Pliegue de Proteína , Agua , Análisis de Inyección de Flujo , Interleucina-1/genética , Interleucina-1/metabolismo , Cinética , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Desnaturalización Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Propiedades de SuperficieRESUMEN
Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J., and Adams, J. A. (1999) Biochemistry 38, 12072-12079). To assess whether specific structural changes are associated with the binding of nucleotides, hydrogen-deuterium exchange experiments were performed on the enzyme in the absence and presence of ADP. Four regions of the protein are protected from exchange in the presence of ADP. Two regions encompass the catalytic and glycine-rich loops and are integral parts of the active site. Conversely, protection of probes in the C terminus is consistent with nucleotide-induced domain closure. One protected probe encompasses a portion of helix C, a secondary structural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of some protein kinases. The combined data suggest that binding of the nucleotide has distal structural effects that may include stabilizing the closed state of the enzyme and altering the position of a critical helix outside the active site. The latter represents the first evidence that the nucleotide alone can induce changes in helix C in solution.
Asunto(s)
Adenosina Difosfato/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Nucleótidos/metabolismo , Animales , Sitios de Unión , Catálisis , Deuterio/metabolismo , Activación Enzimática , Escherichia coli/metabolismo , Glicina/metabolismo , Hidrógeno/metabolismo , Cinética , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Factores de TiempoRESUMEN
Kinetic data measured from folding of the protein interleukin-1ß fits best to three exponential phases when studied with tryptophan fluorescence but only two exponential phases when measured using other methods. The technique of ANS fluorescence was used to determine whether the additional phase observed in tryptophan fluorescence was also detected with ANS dye binding. Unlike trytophan fluorescence, the ANS fluorescence was highly dependent on the concentration of protein present during the folding experiment. Experimental controls provide evidence that ANS binds to protein aggregates, present at higher concentrations and absent at lower concentrations. Protein concentration-dependent folding studies demonstrate that, at lower interleukin-1ß concentrations, tryptophan fluorescence kinetics can be fit adequately with a two exponential fit. This study indicates that (1) measured interleukin-1ß folding kinetics fit to a 2 phase model and (2) at higher protein concentrations, transient association of IL-1ß may result in a kinetic fit of 3 phases.
RESUMEN
A point mutation, lysine 97 to isoleucine, in the all-beta cytokine interleukin-1 beta (IL-1 beta) exhibits an increased propensity to form inclusion bodies in vivo and aggregates in vitro. In an effort to better understand the aggregation reaction and determine when intervention may allow rescue of protein from aggregation during renaturation, we developed a novel application of mass spectrometry using isotopic labeling to determine the step(s) at which K97I commits to either the native or aggregated state. Interestingly, despite the early formation of a folding intermediate ensemble at an observed rate lambda(2) of 4.0 s(-1), K97I commits to folding at a significantly slower rate lambda(CF) of 0.021 s(-1). This rate of commitment to folding is in excellent agreement with the observed rate of K97I native state formation (lambda(1) = 0.018 s(-1)). K97I also commits slowly to aggregation at an observed rate lambda(CA) of 0.023 s(-1). Earlier folding species and aggregates present prior to these commitment steps are likely to be in a reversible equilibrium between monomeric folding intermediates and higher-order oligomers. Kinetic and equilibrium experimental measurements of folding and aggregation processes are consistent with a nucleation-dependent model of aggregation.
Asunto(s)
Interleucina-1/química , Pliegue de Proteína , Animales , Cinética , Espectrometría de MasasRESUMEN
Cyclic AMP dependent protein kinase (PKA) is controlled, in part, by the subcellular localization of the enzyme (). Discovery of dual specificity anchoring proteins (d-AKAPs) indicates that not only is the type II, but also the type I, enzyme localized (). It appears that the type I enzyme is localized in a novel, dynamic fashion as opposed to the apparent static localization of the type II enzyme. Recently, the structure of the dimerization/docking (D/D) domain from the type II enzyme was solved (). This work revealed an X-type four-helix bundle motif with a hydrophobic patch that modulates AKAP interactions. To understand the dynamic versus static localization of PKA, multidimensional NMR techniques were used to investigate the structural features of the type I D/D domain. Our results indicate a conserved helix-turn-helix motif in the type I and type II D/D domains. However, important differences between the two domains are evident in the extreme NH(2) terminus: this region is extended in the type II domain, whereas it is helical in the type I protein. The NH(2)-terminal residues in RIIalpha contain determinants for anchoring, and the orientation and packing of this helical element in the RIalpha structure may have profound consequences in the recognition surface presented to the AKAPs.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/química , Secuencia de Aminoácidos , Cromatografía en Gel , Dicroismo Circular , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Escherichia coli/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The folding of Escherichia coli dihydrofolate reductase was examined at pH 7.8 and 15 degrees C by using stopped-flow fluorescence and absorbance spectroscopies. The formation of a highly fluorescent intermediate occurs with relaxation times ranging between 142 and 343 msec, whereas stopped-flow absorbance spectroscopy using methotrexate binding assays shows a distinct lag phase during these time frames for the native state. The lag in absorbance kinetics and the lack of fast-track folding events indicate that the formation of this ensemble of intermediates is an obligatory step in the folding reaction.
Asunto(s)
Proteínas Bacterianas/química , Tetrahidrofolato Deshidrogenasa/química , Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Metotrexato/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Espectrometría de Fluorescencia , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
The overall structure of the transition-state and intermediate ensembles observed experimentally for dihydrofolate reductase and interleukin-1beta can be obtained by using simplified models that have almost no energetic frustration. The predictive power of these models suggests that, even for these very large proteins with completely different folding mechanisms and functions, real protein sequences are sufficiently well designed, and much of the structural heterogeneity observed in the intermediates and the transition-state ensembles is determined by topological effects.
Asunto(s)
Proteínas Bacterianas/química , Simulación por Computador , Interleucina-1/química , Modelos Moleculares , Tetrahidrofolato Deshidrogenasa/química , Escherichia coli/enzimología , Conformación Proteica , Pliegue de Proteína , TermodinámicaRESUMEN
A point mutation, lysine 97 --> isoleucine (K97I), in a surface loop in the beta-sheet protein interleukin 1beta (IL-1beta), exhibits increased levels of inclusion body (IB) formation relative to the wild-type protein (WT) when expressed in Escherichia coli. Despite the common observation that less stable proteins are often found in IBs, K97I is more stable than WT. We examined the folding pathway of the mutant and wild-type proteins at pH 6.5 and 25 degrees C with manual-mixing and stopped-flow optical spectroscopy to determine whether changes in the properties of transiently populated species in vitro correlate with the observation of increased aggregation in vivo. The refolding reactions of the WT and K97I proteins are both described by three exponential processes. Two exponential processes characterize fast events (0.1-1.0 s) in folding while the third exponential process correlates with a slow (70 s) single pathway to and from the native state. The K97I replacement affects the earlier steps in the refolding pathway. Aggregation, absent in the WT refolding reaction, occurs in K97I above a critical protein concentration of 18 microM. This observation is consistent with an initial nucleation step mediating protein aggregation. Stopped-flow kinetic studies of the K97I aggregation process demonstrate that K97I aggregates most rapidly during the earliest refolding times, when unfolded protein conformers remain highly populated and the concentration of folding intermediates is low. Folding and aggregation studies together support a model in which the formation of stable folding intermediates afford protection against further K97I aggregation.
Asunto(s)
Interleucina-1/química , Interleucina-1/metabolismo , Pliegue de Proteína , Sustitución de Aminoácidos , Clonación Molecular , Estabilidad de Medicamentos , Escherichia coli , Guanidina , Isoleucina , Cinética , Lisina , Sustancias Macromoleculares , Modelos Moleculares , Mutación Puntual , Desnaturalización Proteica , Renaturación de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Dispersión de RadiaciónRESUMEN
Compartmentalization of signal transduction enzymes into signaling complexes is an important mechanism to ensure the specificity of intracellular events. Formation of these complexes is mediated by specialized protein motifs that participate in protein-protein interactions. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) is localized through interaction of the regulatory (R) subunit dimer with A-kinase-anchoring proteins (AKAPs). We now report the solution structure of the type II PKA R-subunit fragment RIIalpha(1-44), which encompasses both the AKAP-binding and dimerization interfaces. This structure incorporates an X-type four-helix bundle dimerization motif with an extended hydrophobic face that is necessary for high-affinity AKAP binding. NMR data on the complex between RIIalpha(1-44) and an AKAP fragment reveals extensive contacts between the two proteins. Interestingly, this same dimerization motif is present in other signaling molecules, the S100 family. Therefore, the X-type four-helix bundle may represent a conserved fold for protein-protein interactions in signal transduction.