RESUMEN
Integral membrane proteins (IMPs) play central roles in cellular physiology and represent the majority of known drug targets. Single-molecule fluorescence and fluorescence resonance energy transfer (FRET) methods have recently emerged as valuable tools for investigating structure-function relationships in IMPs. This review focuses on the practical foundations required for examining polytopic IMP function using single-molecule FRET (smFRET) and provides an overview of the technical and conceptual frameworks emerging from this area of investigation. In this context, we highlight the utility of smFRET methods to reveal transient conformational states critical to IMP function and the use of smFRET data to guide structural and drug mechanism-of-action investigations. We also identify frontiers where progress is likely to be paramount to advancing the field.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Proteínas de la Membrana , Imagen Individual de Molécula , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Transferencia Resonante de Energía de Fluorescencia/métodos , Imagen Individual de Molécula/métodos , Humanos , AnimalesRESUMEN
Atomic force microscopy has emerged as a valuable complementary technique in membrane structural biology. The apparatus is capable of probing individual membrane proteins in fluid lipid bilayers at room temperature with spatial resolution at the molecular length scale. Protein conformational dynamics are accessible over a range of biologically relevant timescales. This chapter presents methodology our group uses to achieve robust AFM image data of the General Secretory system, the primary pathway of protein export from the cytoplasm to the periplasm of E. coli. Emphasis is given to measuring and maintaining biochemical activity and to objective AFM image processing methods. For example, the biochemical assays can be used to determine chemomechanical coupling efficiency of surface adsorbed translocases. The Hessian blob algorithm and its extension to nonlocalized linear features, the line detection algorithm, provide automated feature delineations. Many of the methods discussed here can be applied to other membrane protein systems of interest.
Asunto(s)
Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Imagen Individual de Molécula/métodos , Algoritmos , Citoplasma/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Microscopía de Fuerza Atómica , Periplasma/metabolismo , Conformación Proteica , Transporte de ProteínasRESUMEN
Surface-supported lipid bilayers are used widely throughout the nanoscience community as cellular membrane mimics. For example, they are frequently employed in single-molecule atomic force microscopy (AFM) studies to shed light on membrane protein conformational dynamics and folding. However, in AFM as well as in other surface-sensing techniques, the close proximity of the supporting surface raises questions about preservation of the biochemical activity. Employing the model translocase from the general secretory (Sec) system of Escherichia coli, here we quantify the activity via two biochemical assays in surface-supported bilayers. The first assesses ATP hydrolysis and the second assesses polypeptide translocation across the membrane via protection from added protease. Hydrolysis assays revealed distinct levels of activation ranging from medium (translocase-activated) to high (translocation-associated) that were similar to traditional solution experiments and further identified an adenosine triphosphatase population exhibiting characteristics of conformational hysteresis. Translocation assays revealed turn over numbers that were comparable to solution but with a 10-fold reduction in apparent rate constant. Despite differences in kinetics, the chemomechanical coupling (ATP hydrolyzed per residue translocated) only varied twofold on glass compared to solution. The activity changed with the topographic complexity of the underlying surface. Rough glass coverslips were favored over atomically flat mica, likely due to differences in frictional coupling between the translocating polypeptide and surface. Neutron reflectometry and AFM corroborated the biochemical measurements and provided structural characterization of the submembrane space and upper surface of the bilayer. Overall, the translocation activity was maintained for the surface-adsorbed Sec system, albeit with a slower rate-limiting step. More generally, polypeptide translocation activity measurements yield valuable quantitative metrics to assess the local environment about surface-supported lipid bilayers.
Asunto(s)
Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Adenosina Trifosfato/metabolismo , Activación Enzimática , Translocasas Mitocondriales de ADP y ATP/metabolismo , Transporte de Proteínas , Propiedades de SuperficieRESUMEN
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
The electronic band structure of twisted bilayer graphene develops van Hove singularities whose energy depends on the twist angle between the two layers. Using Raman spectroscopy, we monitor the evolution of the electronic band structure upon doping using the G peak area which is enhanced when the laser photon energy is resonant with the energy separation of the van Hove singularities. Upon charge doping, the Raman G peak area initially increases for twist angles larger than a critical angle and decreases for smaller angles. To explain this behavior with twist angle, the energy separation of the van Hove singularities must decrease with increasing charge density demonstrating the ability to modify the electronic and optical properties of twisted bilayer graphene with doping.
RESUMEN
Raman spectroscopy, a fast and nondestructive imaging method, can be used to monitor the doping level in graphene devices. We fabricated chemical vapor deposition (CVD) grown graphene on atomically flat hexagonal boron nitride (hBN) flakes and SiO2 substrates. We compared their Raman response as a function of charge carrier density using an ion gel as a top gate. The G peak position, the 2D peak position, the 2D peak width and the ratio of the 2D peak area to the G peak area show a dependence on carrier density that differs for hBN compared to SiO2. Histograms of two-dimensional mapping are used to compare the fluctuations in the Raman peak properties between the two substrates. The hBN substrate has been found to produce fewer fluctuations at the same charge density owing to its atomically flat surface and reduced charged impurities.