RESUMEN
Strand-separation is emerging as a novel DNA recognition mechanism but the underlying mechanisms and quantitative contribution of strand-separation to fidelity remain obscure. The bacterial DNA adenine methyltransferase, CcrM, recognizes 5'GANTC'3 sequences through a DNA strand-separation mechanism with unusually high selectivity. To explore this novel recognition mechanism, we incorporated Pyrrolo-dC into cognate and noncognate DNA to monitor the kinetics of strand-separation and used tryptophan fluorescence to follow protein conformational changes. Both signals are biphasic and global fitting showed that the faster phase of DNA strand-separation was coincident with the protein conformational transition. Non-cognate sequences did not display strand-separation and methylation was reduced > 300-fold, providing evidence that strand-separation is a major determinant of selectivity. Analysis of an R350A mutant showed that the enzyme conformational step can occur without strand-separation, so the two events are uncoupled. A stabilizing role for the methyl-donor (SAM) is proposed; the cofactor interacts with a critical loop which is inserted between the DNA strands, thereby stabilizing the strand-separated conformation. The results presented here are broadly applicable to the study of other N6-adenine methyltransferases that contain the structural features implicated in strand-separation, which are found widely dispersed across many bacterial phyla, including human and animal pathogens, and some Eukaryotes.
Asunto(s)
ADN , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica) , Humanos , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , ADN/química , Metilación de ADN , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Adenina/metabolismo , Cinética , Especificidad por SustratoRESUMEN
The ability to monitor drugs, metabolites, hormones, and other biomarkers in situ in the body would greatly advance both clinical practice and biomedical research. To this end, we are developing electrochemical aptamer-based (EAB) sensors, a platform technology able to perform real-time, in vivo monitoring of specific molecules irrespective of their chemical or enzymatic reactivity. An important obstacle to the deployment of EAB sensors in the challenging environments found in the living body is signal drift, whereby the sensor signal decreases over time. To date, we have demonstrated a number of approaches by which this drift can be corrected sufficiently well to achieve good measurement precision over multihour in vivo deployments. To achieve a much longer in vivo measurement duration, however, will likely require that we understand and address the sources of this effect. In response, here, we have systematically examined the mechanisms underlying the drift seen when EAB sensors and simpler, EAB-like devices are challenged in vitro at 37 °C in whole blood as a proxy for in vivo conditions. Our results demonstrate that electrochemically driven desorption of a self-assembled monolayer and fouling by blood components are the two primary sources of signal loss under these conditions, suggesting targeted approaches to remediating this degradation and thus improving the stability of EAB sensors and other, similar electrochemical biosensor technologies when deployed in the body.
RESUMEN
The physics of proteins interacting with surfaces can differ significantly from those seen when the same proteins are free in bulk solution. As an example, we describe here the extent to which site-specific attachment to a chemically well-defined macroscopic surface alters the ability of several stabilizing and destabilizing cosolutes to modulate protein folding thermodynamics. We determined this via guanidinium denaturations performed in the presence of varying concentrations of cosolutes when proteins were either site-specifically attached to self-assembled monolayers on gold or free in bulk solution. Doing this we found that the extent to which guanidinium (a destabilizing Hofmeister cation), sulfate (a stabilizing Hofmeister anion), and urea (a neutral denaturant) alter the folding free energy remains indistinguishable whether proteins are surface-attached or free in bulk solution. In sharp contrast, however, neutral osmolytes sucrose and glycerol, which significantly stabilize proteins in bulk solution, do not measurably affect their stability when they are attached to a hydroxyl-terminated surface. In contrast, we recovered bulk solution-like stabilization when the attachment surface was instead carboxyl-terminated. It thus appears that chemistry-specific surface interactions can dramatically alter the way in which biomolecules interact with other components of the system.
Asunto(s)
Radical Hidroxilo/química , Polímeros/química , Proteínas/química , Guanidina/química , Pliegue de Proteína , Sulfatos/química , Propiedades de Superficie , Termodinámica , Urea/químicaRESUMEN
The ability to monitor drug and biomarker concentrations in the body with high frequency and in real time would revolutionize our understanding of biology and our capacity to personalize medicine. The few inâ vivo molecular sensors that currently exist, however, all rely on the specific chemical or enzymatic reactivity of their targets and thus are not generalizable. In response, we demonstrate here an electrochemical sensing architecture based on binding-induced protein folding that is 1)â independent of the reactivity of its targets, 2)â reagentless, real-time, and with a resolution of seconds, and 3)â selective enough to deploy in undiluted bodily fluids. As a proof of principle, we use the SH3 domain from human Fyn kinase to build a sensor that discriminates between the protein's peptide targets and responds rapidly and quantitatively even when challenged in whole blood. The resulting sensor architecture could drastically expand the chemical space accessible to continuous, real-time biosensors.
Asunto(s)
Técnicas Biosensibles/métodos , Proteínas Proto-Oncogénicas c-fyn/química , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Técnicas Electroquímicas , Electrodos , Oro/química , Humanos , Concentración Osmolar , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Pliegue de Proteína , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Dominios Homologos srcRESUMEN
The electrochemical aptamer-based (E-AB) sensing platform appears to be a convenient (rapid, single-step, and calibration-free) and modular approach to measure concentrations of specific molecules (irrespective of their chemical reactivity) directly in blood and even in situ in the living body. Given these attributes, the platform may thus provide significant opportunities to render therapeutic drug monitoring (the clinical practice in which dosing is adjusted in response to plasma drug measurements) as frequent and convenient as the measurement of blood sugar has become for diabetics. The ability to measure arbitrary molecules in the body in real time could even enable closed-loop feedback control over plasma drug levels in a manner analogous to the recently commercialized controlled blood sugar systems. As initial exploration of this, we describe here the selection of an aptamer against vancomycin, a narrow therapeutic window antibiotic for which therapeutic monitoring is a critical part of the standard of care, and its adaptation into an electrochemical aptamer-based (E-AB) sensor. Using this sensor, we then demonstrate: (i) rapid (seconds) and convenient (single-step and calibration-free) measurement of plasma vancomycin in finger-prick-scale samples of whole blood, (ii) high-precision measurement of subject-specific vancomycin pharmacokinetics (in a rat animal model), and (iii) high-precision, closed-loop feedback control over plasma levels of the drug (in a rat animal model). The ability to not only track (with continuous-glucose-monitor-like measurement frequency and convenience) but also actively control plasma drug levels provides an unprecedented route toward improving therapeutic drug monitoring and, more generally, the personalized, high-precision delivery of pharmacological interventions.
Asunto(s)
Antibacterianos/sangre , Aptámeros de Nucleótidos/química , Monitoreo de Drogas/métodos , Técnicas Electroquímicas/métodos , Vancomicina/sangre , Animales , Antibacterianos/química , Bovinos , Masculino , Ratas Sprague-Dawley , Vancomicina/químicaRESUMEN
Electrochemical sensors are major players in the race for improved molecular diagnostics due to their convenience, temporal resolution, manufacturing scalability, and their ability to support real-time measurements. This is evident in the ever-increasing number of health-related electrochemical sensing platforms, ranging from single-measurement point-of-care devices to wearable devices supporting immediate and continuous monitoring. In support of the need for such systems to rapidly process large data volumes, we describe here an open-source, easily customizable, multiplatform compatible program for the real-time control, processing, and visualization of electrochemical data. The software's architecture is modular and fully documented, allowing the easy customization of the code to support the processing of voltammetric (e.g., square-wave and cyclic) and chronoamperometric data. The program, which we have called Software for the Analysis and Continuous Monitoring of Electrochemical Systems (SACMES), also includes a graphical interface allowing the user to easily change analysis parameters (e.g., signal/noise processing, baseline correction) in real-time. To demonstrate the versatility of SACMES we use it here to analyze the real-time data output by (1) the electrochemical, aptamer-based measurement of a specific small-molecule target, (2) a monoclonal antibody-detecting DNA-scaffold sensor, and (3) the determination of the folding thermodynamics of an electrode-attached, redox-reporter-modified protein.
Asunto(s)
Electroquímica/métodos , Programas Informáticos , Animales , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Técnicas Biosensibles , ADN/análisis , Procesamiento Automatizado de Datos , Masculino , Pliegue de Proteína , Ratas , Ratas Sprague-Dawley , Relación Señal-Ruido , Factores de TiempoRESUMEN
The faster a disease can be diagnosed, the sooner effective treatment can be initiated, motivating a drive to replace standard laboratory techniques with point-of-care technologies that return answers in minutes rather than hours. Thus motivated, we describe the development of an E-DNA scaffold sensor for the rapid and convenient measurement of antibodies diagnostic of syphilis. To achieve this (and in contrast to previous sensors of this class, which relied on single, linear epitopes for detection), we utilized a near full-length antigen as the sensor's recognition element, allowing us to simultaneously display multiple epitopes. The resultant sensor is able to detect antibodies against Treponema pallidum pallidum, the causative agent of syphilis, at clinically relevant concentrations in samples in less than 10 min. Preliminary results obtained using sero-positive and sero-negative human samples suggest the clinical sensitivity and specificity of the approach compare well to current gold-standard tests, while being simple and rapid enough to deploy at the point of care.
Asunto(s)
Anticuerpos Antibacterianos/sangre , ADN/química , Sífilis/diagnóstico , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Secuencia de Bases , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Escherichia coli/genética , Humanos , Ácidos Nucleicos Inmovilizados/química , Azul de Metileno/química , Oxidación-Reducción , Treponema pallidum/químicaRESUMEN
Despite the importance of protein-surface interactions in both biology and biotechnology, our understanding of their origins is limited due to a paucity of experimental studies of the thermodynamics behind such interactions. In response, we have characterized the extent to which interaction with a chemically well-defined macroscopic surface alters the stability of proteinâ L. To do so, we site-specifically attached a redox-reporter-modified protein variant to a hydroxy-terminated monolayer on a gold surface and then used electrochemistry to monitor its guanidine denaturation and determine its folding free energy. Comparison with the free energy seen in solution indicates that interaction with this surface stabilizes the protein by 6â kJ mol-1 , a value that is in good agreement with theoretical estimates of the entropic consequences of surface-induced excluded volume effects, thus suggesting that chemically specific interactions with this surface (e.g., electrostatic interactions) are limited in magnitude.
Asunto(s)
Oro/química , Proteínas/química , Termodinámica , Técnicas Electroquímicas , Pliegue de Proteína , Estabilidad Proteica , Electricidad Estática , Propiedades de SuperficieRESUMEN
Two DNA methyltransferases, Dam and ß-class cell cycle-regulated DNA methyltransferase (CcrM), are key mediators of bacterial epigenetics. CcrM from the bacterium Caulobacter crescentus (CcrM C. crescentus, methylates adenine at 5'-GANTC-3') displays 105-107-fold sequence discrimination against noncognate sequences. However, the underlying recognition mechanism is unclear. Here, CcrM C. crescentus activity was either improved or mildly attenuated with substrates having one to three mismatched bp within or adjacent to the recognition site, but only if the strand undergoing methylation is left unchanged. By comparison, single-mismatched substrates resulted in up to 106-fold losses of activity with α (Dam) and γ-class (M.HhaI) DNA methyltransferases. We found that CcrM C. crescentus has a greatly expanded DNA-interaction surface, covering six nucleotides on the 5' side and eight nucleotides on the 3' side of its recognition site. Such a large interface may contribute to the enzyme's high sequence fidelity. CcrM C. crescentus displayed the same sequence discrimination with single-stranded substrates, and a surprisingly large (>107-fold) discrimination against ssRNA was largely due to the presence of two or more riboses within the cognate (DNA) site but not outside the site. Results from C-terminal truncations and point mutants supported our hypothesis that the recently identified C-terminal, 80-residue segment is essential for dsDNA recognition but is not required for single-stranded substrates. CcrM orthologs from Agrobacterium tumefaciens and Brucella abortus share some of these newly discovered features of the C. crescentus enzyme, suggesting that the recognition mechanism is conserved. In summary, CcrM C. crescentus uses a previously unknown DNA recognition mechanism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/enzimología , ADN Bacteriano/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Agrobacterium tumefaciens/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Disparidad de Par Base , Brucella abortus/enzimología , Dominio Catalítico , Metilación de ADN , ADN Bacteriano/genética , Dominios Proteicos , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/químicaRESUMEN
α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula.
Asunto(s)
Amiloide/química , Benzoxazoles/química , Benzoxazoles/farmacología , Agregado de Proteínas/efectos de los fármacos , alfa-Sinucleína/química , Amiloide/antagonistas & inhibidores , Amiloide/ultraestructura , Transferencia Resonante de Energía de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Conformación Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/antagonistas & inhibidores , alfa-Sinucleína/ultraestructuraRESUMEN
Whereas proteins generally remain stable upon interaction with biological surfaces, they frequently unfold on and adhere to artificial surfaces. Understanding the physicochemical origins of this discrepancy would facilitate development of protein-based sensors and other technologies that require surfaces that do not compromise protein structure and function. To date, however, only a small number of such artificial surfaces have been reported, and the physics of why these surfaces support functional biomolecules while others do not has not been established. Thus motivated, we have developed an electrochemical approach to determining the folding free energy of proteins site-specifically attached to chemically well-defined, macroscopic surfaces. Comparison with the folding free energies seen in bulk solution then provides a quantitative measure of the extent to which surface interactions alter protein stability. As proof-of-principle, we have characterized the FynSH3 domain site-specifically attached to a hydroxyl-coated surface. Upon guanidinium chloride denaturation, the protein unfolds in a reversible, two-state manner with a free energy within 2 kJ/mol of the value seen in bulk solution. Assuming that excluded volume effects stabilize surface-attached proteins, this observation suggests there are countervening destabilizing interactions with the surface that, under these conditions, are similar in magnitude. Our technique constitutes an unprecedented experimental tool with which to answer long-standing questions regarding the molecular-scale origins of protein-surface interactions and to facilitate rational optimization of surface biocompatibility.
Asunto(s)
Pliegue de Proteína , Proteínas Proto-Oncogénicas c-fyn/química , Termodinámica , Dominios Homologos src , Técnicas Electroquímicas , Humanos , Estabilidad ProteicaRESUMEN
Here we demonstrate a new class of reagentless, single-step sensors for the detection of proteins and peptides that is the electrochemical analog of fluorescence polarization (fluorescence anisotropy), a versatile optical approach widely employed to this same end. Our electrochemical sensors consist of a redox-reporter-modified protein (the "receptor") site-specifically anchored to an electrode via a short, flexible polypeptide linker. Interaction of the receptor with its binding partner alters the efficiency with which the reporter approaches the electrode surface, thus causing a change in redox current upon voltammetric interrogation. As our first proof-of-principle we employed the bacterial chemotaxis protein CheY as our receptor. Interaction with either of CheY's two binding partners, the P2 domain of the chemotaxis kinase, CheA, or the 16-residue "target region" of the flagellar switch protein, FliM, leads to easily measurable changes in output current that trace Langmuir isotherms within error of those seen in solution. Phosphorylation of the electrode-bound CheY decreases its affinity for CheA-P2 and enhances its affinity for FliM in a manner likewise consistent with its behavior in solution. As expected given the proposed sensor signaling mechanism, the magnitude of the binding-induced signal change depends on the placement of the redox reporter on the receptor. Following these preliminary studies with CheY, we also developed and characterized additional sensors aimed at the detection of specific antibodies using the relevant protein antigens as the receptor. These exhibit excellent detection limits for their targets without the use of reagents or wash steps. This novel, protein-based electrochemical sensing architecture provides a new and potentially promising approach to sensors for the single-step measurement of specific proteins and peptides.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas/instrumentación , Proteínas Quimiotácticas Aceptoras de Metilo/química , Proteínas de Escherichia coli , Indicadores y Reactivos/química , Proteínas Quimiotácticas Aceptoras de Metilo/análisisRESUMEN
The efficiency with which square-wave voltammetry differentiates faradic and charging currents makes it a particularly sensitive electroanalytical approach, as evidenced by its ability to measure nanomolar or even picomolar concentrations of electroactive analytes. Because of the relative complexity of the potential sweep it uses, however, the extraction of detailed kinetic and mechanistic information from square-wave data remains challenging. In response, we demonstrate here a numerical approach by which square-wave data can be used to determine electron transfer rates. Specifically, we have developed a numerical approach in which we model the height and the shape of voltammograms collected over a range of square-wave frequencies and amplitudes to simulated voltammograms as functions of the heterogeneous rate constant and the electron transfer coefficient. As validation of the approach, we have used it to determine electron transfer kinetics in both freely diffusing and diffusionless surface-tethered species, obtaining electron transfer kinetics in all cases in good agreement with values derived using non-square-wave methods.
Asunto(s)
Electrones , Transporte de Electrón , CinéticaRESUMEN
The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the κ opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble ß-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.
Asunto(s)
Dinorfinas/metabolismo , Modelos Moleculares , Receptores Opioides kappa/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Dinorfinas/química , Espectroscopía de Resonancia por Spin del Electrón , Estudios de Factibilidad , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Ligandos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Opioides kappa/química , Receptores Opioides kappa/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Electricidad Estática , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Andamios del Tejido/químicaRESUMEN
The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the ß-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120-H43-T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cu-bound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?
Asunto(s)
Superóxido Dismutasa/química , Sustitución de Aminoácidos , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Dominio Catalítico/genética , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Evolución Molecular , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Simulación de Dinámica Molecular , Movimiento (Física) , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ(lag)) and maximum growth rate (ν(max)) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ(lag)/∂[urea] and ∂ log ν(max)/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.
Asunto(s)
Pliegue de Proteína , Precursores de Proteínas/metabolismo , Superóxido Dismutasa/metabolismo , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Precursores de Proteínas/química , Superóxido Dismutasa/química , Superóxido Dismutasa-1 , Urea/químicaRESUMEN
Surface charges of proteins have in several cases been found to function as "structural gatekeepers," which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Pliegue de Proteína , Thermus thermophilus/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Proteína S6 Ribosómica/química , Proteína S6 Ribosómica/metabolismo , Solubilidad , Soluciones , Electricidad Estática , TermodinámicaRESUMEN
Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric ß-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent ß-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in ß-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.