Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles

AbstractTo better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly

A Man-Made ATP-Binding Protein Evolved Independent of Nature Causes Abnormal Growth in Bacterial Cells

Recent advances in de novo protein evolution have made it possible to create synthetic proteins from unbiased libraries that fold into stable tertiary structures with predefined functions. However, it is not known whether such proteins will be functional when expressed inside living cells or how a host organism would respond to an encounter with a non-biological protein. Here, we examine the physiology and morphology of Escherichia coli cells engineered to express a synthetic ATP-binding protein evolved entirely from non-biological origins. We show that this man-made protein disrupts the normal energetic balance of the cell by altering the levels of intracellular ATP. This disruption cascades into a series of events that ultimately limit reproductive competency by inhibiting cell division. We now describe a detailed investigation into the synthetic biology of this man-made protein in a living bacterial organism, and the effect that this protein has on normal cell physiology

Single Particle Difraction at FLASH

Single-pulse coherent diffraction patterns have been collected from randomly injected single particles with a soft X-ray free-electron laser (FEL). The intense focused FEL pulse gives a high-resolution low-noise coherent diffraction pattern of the object before that object turns into a plasma and explodes. A diffraction pattern of a single particle will only be recorded when the particle arrival into the FEL interaction region coincides with FEL pulse arrival and detector integration. The properties of the experimental apparatus coinciding with these three events set the data acquisition rate. For our single particle FLASH diffraction imaging experiments: (1) an aerodynamic lens stack prepared a particle beam that consisted of particles moving at 150-200 m/s positioned randomly in space and time, (2) the 10 fs long FEL pulses were delivered at a fixed rate, and (3) the detector was set to integrate and readout once every two seconds. The effect of these experimental parameters on the rate of data acquisition using randomly injected particles will be discussed. Overall, the ultrashort FEL pulses do not set the limit of the data acquisition, more important is the effective interaction time of the particle crossing the FEL focus, the pulse sequence structure and the detector readout rate. Example diffraction patterns of randomly injected ellipsoidal iron oxide nanoparticles in different orientations are presented. This is the first single particle diffraction data set of identical particles in different orientations collected on a shot-to-shot basis. This data set will be used to test algorithms for recovering 3D structure from single particle diffraction

Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals

Garotas de loja, história social e teoria social [Shop Girls, Social History and Social Theory]

Shop workers, most of them women, have made up a significant proportion of Britain’s labour force since the 1850s but we still know relatively little about their history. This article argues that there has been a systematic neglect of one of the largest sectors of female employment by historians and investigates why this might be. It suggests that this neglect is connected to framings of work that have overlooked the service sector as a whole as well as to a continuing unease with the consumer society’s transformation of social life. One element of that transformation was the rise of new forms of aesthetic, emotional and sexualised labour. Certain kinds of ‘shop girls’ embodied these in spectacular fashion. As a result, they became enduring icons of mass consumption, simultaneously dismissed as passive cultural dupes or punished as powerful agents of cultural destruction. This article interweaves the social history of everyday shop workers with shifting representations of the ‘shop girl’, from Victorian music hall parodies, through modernist social theory, to the bizarre bombing of the Biba boutique in London by the Angry Brigade on May Day 1971. It concludes that progressive historians have much to gain by reclaiming these workers and the service economy that they helped create