RESUMEN
Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Unión al Calcio/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Membrana Dobles de Lípidos/química , Proteínas de Transporte de Monosacáridos/química , Proteínas de Unión Periplasmáticas/química , Precursores de Proteínas/química , Proteína SecA/química , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Membrana Dobles de Lípidos/metabolismo , Microscopía de Fuerza Atómica , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Unión Proteica , Multimerización de Proteína , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Proteolípidos/química , Proteolípidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Translocación SEC/química , Canales de Translocación SEC/genética , Canales de Translocación SEC/metabolismo , Proteína SecA/genética , Proteína SecA/metabolismoRESUMEN
SecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in Escherichia coli. This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contribute to this motion during the transition state of the ATP hydrolysis cycle.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Canales de Translocación SEC/química , Canales de Translocación SEC/metabolismo , Análisis de la Célula Individual/métodos , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/efectos de los fármacos , Escherichia coli , Hidrólisis , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Canales de Translocación SEC/efectos de los fármacos , Proteína SecARESUMEN
Imaging by atomic force microscopy (AFM) offers high-resolution descriptions of many biological systems; however, regardless of resolution, conclusions drawn from AFM images are only as robust as the analysis leading to those conclusions. Vital to the analysis of biomolecules in AFM imagery is the initial detection of individual particles from large-scale images. Threshold and watershed algorithms are conventional for automatic particle detection but demand manual image preprocessing and produce particle boundaries which deform as a function of user-defined parameters, producing imprecise results subject to bias. Here, we introduce the Hessian blob to address these shortcomings. Combining a scale-space framework with measures of local image curvature, the Hessian blob formally defines particle centers and their boundaries, both to subpixel precision. Resulting particle boundaries are independent of user defined parameters, with no image preprocessing required. We demonstrate through direct comparison that the Hessian blob algorithm more accurately detects biomolecules than conventional AFM particle detection techniques. Furthermore, the algorithm proves largely insensitive to common imaging artifacts and noise, delivering a stable framework for particle analysis in AFM.
RESUMEN
Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on microscope cover glass following a straight-forward cleaning procedure. Direct comparison between AFM data obtained on glass and on mica substrates show no major differences in image fidelity. Repeated association of the ATPase SecA with the cytoplasmic protrusion of SecYEG demonstrates that the translocon remains competent for binding after tens of minutes of continuous AFM imaging. This opens the door for precision long-timescale investigations of the active translocase in near-native conditions and, more generally, for integration of high resolution biological AFM with many powerful optical techniques that require non-birefringent substrates.