The emergence of COVID-19 has presented employees and employers new challenges as many employees and managers were forced to work in a remote environment for the first time. For many reasons, ...managing virtual teams is different than managing employees in a traditional face-to-face office environment. Although many managers have been learning how to lead their virtual teams over the last several months, we offer five steps for leaders to follow for how to maximize the effectiveness of a remote workplace. By taking specific actions and ensuring the organization has a culture to support their virtual workforce, leaders can improve the performance output and engagement of their teams. The five steps are: first establish and explain the new reality; second, establish and maintain a culture of trust; third, upgrade leadership communication tools and techniques to better inform virtual employees; fourth, encourage shared leadership among team members; and fifth, to create and periodically perform alignment audits to ensure virtual employees are aligned with the organization’s cultural values including its commitment to mission. All these steps start with the realization that managing a team is going to be different when the members are dispersed, and new leadership strategies, communication routines and tools are required.
ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their ...large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.
ABSTRACT
ATP‐binding cassette (ABC) transporters are ubiquitous across all realms of life. Dogma suggests that bacterial ABC transporters include both importers and exporters, whilst eukaryotic ...members of this family are solely exporters, implying that ABC import function was lost during evolution. This view is being challenged, for example energy‐coupling factor (ECF)‐type ABC importers appear to fulfil important roles in both algae and plants where they form the ABCI sub‐family. Herein we discuss whether bacterial Type I and Type II ABC importers also made the transition into extant eukaryotes. Various studies suggest that Type I importers exist in algae and the liverwort family of primitive non‐vascular plants, but not in higher plants. The existence of eukaryotic Type II importers is also supported: a transmembrane protein homologous to vitamin B12 import system transmembrane protein (BtuC), hemin transport system transmembrane protein (HmuU) and high‐affinity zinc uptake system membrane protein (ZnuB) is present in the Cyanophora paradoxa genome. This protein has homologs within the genomes of red algae. Furthermore, its candidate nucleotide‐binding domain (NBD) shows closest similarity to other bacterial Type II importer NBDs such as BtuD. Functional studies suggest that Type I importers have roles in maintaining sulphate levels in the chloroplast, whilst Type II importers probably act as importers of Mn2+ or Zn2+, as inferred by comparisons with bacterial homologs. Possible explanations for the presence of these transporters in simple plants, but not in other eukaryotic organisms, are considered. In order to utilise the existing nomenclature for eukaryotic ABC proteins, we propose that eukaryotic Type I and II importers be classified as ABCJ and ABCK transporters, respectively.
Cryo-electron microscopy of membrane proteins. Electrons pass through the sample and are focused and imaged in the transmission electron microscope. The sample of membrane proteins is encapsulated in ...a microscopically thin layer of glassy ice. In most cases, free detergent micelles will be present as well as protein/detergent complexes.
Display omitted
•Cryo-electron microscopy applied to membrane proteins.•Different detergents can be tuned to the methodology.•Weak contrast and the confounding effect of the micelle/nanodisc.•Interpretation of medium resolution structures.
Membrane proteins represent a large proportion of the proteome, but have characteristics that are problematic for many methods in modern molecular biology (that have often been developed with soluble proteins in mind). For structural studies, low levels of expression and the presence of detergent have been thorns in the flesh of the membrane protein experimentalist. Here we discuss the use of cryo-electron microscopy in breakthrough studies of the structures of membrane proteins. This method can cope with relatively small quantities of sample and with the presence of detergent. Until recently, cryo-electron microscopy could not deliver high-resolution structures of membrane proteins, but recent developments in transmission electron microscope technology and in the image processing of single particles imaged in the microscope have revolutionized the field, allowing high resolution structures to be obtained. Here we focus on the specific issues surrounding the application of cryo-electron microscopy to the study of membrane proteins, especially in the choice of a system to keep the protein soluble.
The classic conceptualization of ATP binding cassette (ABC) transporter function is an ATP‐dependent conformational change coupled to transport of a substrate across a biological membrane via the ...transmembrane domains (TMDs). The binding of two ATP molecules within the transporter's two nucleotide binding domains (NBDs) induces their dimerization. Despite retaining the ability to bind nucleotides, isolated NBDs frequently fail to dimerize. ABC proteins without a TMD, for example ABCE and ABCF, have NBDs tethered via elaborate linkers, further supporting that NBD dimerization does not readily occur for isolated NBDs. Intriguingly, even in full‐length transporters, the NBD‐dimerized, outward‐facing state is not as frequently observed as might be expected. This leads to questions regarding what drives NBD interaction and the role of the TMDs or linkers. Understanding the NBD–nucleotide interaction and the subsequent NBD dimerization is thus pivotal for understanding ABC transporter activity in general. Here, we hope to provide new insights into ABC protein function by discussing the perplexing issue of (missing) NBD dimerization in isolation and in the context of full‐length ABC proteins.
ATP-binding cassette (ABC) transporters are membrane proteins present in all kingdoms of life. We have considered the disordered region that connects the N- and C-terminal halves in many eukaryotic ...ABC transporters, allowing all four consensus functional domains to be linked. The recent availability of structures of ABC transporters containing linker regions has allowed us to identify the start and end points of the connectors as well as hinting at their localisation. We address questions such as: Where did the linker regions come from? Why do some ABC transporters have connectors and others not? What are the rules and roles of the linker regions? What are the consequences of mutations in these connector regions for disease in humans?
The ABC proteins are essential in all living organisms and are mostly transporters of substances across biological membranes.Some eukaryotic ABC transporters have disordered linker regions that join their N- and C-terminal halves together; perhaps as an adaptation to eukaryotic complexity.The evolutionary origins of eukaryotic ABC transporters may provide insight about where the linker regions came from and their functions.The linker region may possess a number of functions and may be regulated by post-translational modifications, such as phosphorylation.Mutations to the linker regions have been observed in a number of human diseases.
The impressive growth in web-mediated organizational relationships has created an escalating interest in how to manage virtual teams successfully. As organizations increasingly expect their managers ...to lead employees in these online groupings, it becomes imperative to identify and train them in the skills to do this effectively. The purpose of this article is to organize and present strategies that organizations have found successful in helping their managers lead virtual teams. While all successful managers must ensure that they have provided the basic organizational support for their employees, especially effective leaders also ensure they build trustworthy relationships. Thus, we emphasize how each strategy contributes to building and sustaining a climate of trust in virtual teams.
Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under ...extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)–PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions −1 and −3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.
We present a new framework to characterize the occurrence rates of planet candidates identified by Kepler based on hierarchical Bayesian modeling, approximate Bayesian computing (ABC), and sequential ...importance sampling. For this study, we adopt a simple 2D grid in planet radius and orbital period as our model and apply our algorithm to estimate occurrence rates for Q1-Q16 planet candidates orbiting solar-type stars. We arrive at significantly increased planet occurrence rates for small planet candidates (Rp < 1.25 R⊕) at larger orbital periods (P > 80 day) compared to the rates estimated by the more common inverse detection efficiency method (IDEM). Our improved methodology estimates that the occurrence rate density of small planet candidates in the habitable zone of solar-type stars is per factor of 2 in planet radius and orbital period. Additionally, we observe a local minimum in the occurrence rate for strong planet candidates marginalized over orbital period between 1.5 and 2 R⊕ that is consistent with previous studies. For future improvements, the forward modeling approach of ABC is ideally suited to incorporating multiple populations, such as planets, astrophysical false positives, and pipeline false alarms, to provide accurate planet occurrence rates and uncertainties. Furthermore, ABC provides a practical statistical framework for answering complex questions (e.g., frequency of different planetary architectures) and providing sound uncertainties, even in the face of complex selection effects, observational biases, and follow-up strategies. In summary, ABC offers a powerful tool for accurately characterizing a wide variety of astrophysical populations.
PDF
