Jacopo Marino

Ten simple rules for getting help from Online Scientific Communities

The increasing complexity of research requires scientists to work at the intersection of multiple fields and to face problems for which their formal education has not prepared them. For example, biologists with no or little background in programming are now often using complex scripts to handle the results from their experiments; vice versa, programmers wishing to enter the world of bioinformatics must know about biochemistry, genetics, and other fields. In this context, communication tools such as mailing lists, web forums, and online communities acquire increasing importance. These tools permit scientists to quickly contact people skilled in a specialized field. A question posed properly to the right online scientific community can help in solving difficult problems, often faster than screening literature or writing to publication authors. The growth of active online scientific communities, such as those listed in Table S1, demonstrates how these tools are becoming an important source of support for an increasing number of researchers. Nevertheless, making proper use of these resources is not easy. Adhering to the social norms of World Wide Web communication—loosely termed “netiquette”—is both important and non-trivial. In this article, we take inspiration from our experience on Internet-shared scientific knowledge, and from similar documents such as “Asking the Questions the Smart Way” and “Getting Answers”, to provide guidelines and suggestions on how to use online communities to solve scientific problems.

Topogenesis of heterologously expressed fragments of the human Y4 GPCR

Fragments of large membrane proteins have the potential to facilitate structural analysis by NMR, but their folding state remains a concern. Here we determined the quality of folding upon heterologous expression for a series of N- or C-terminally truncated fragments of the human Y4 G-protein coupled receptor, amounting to six different complementation pairs. As the individual fragments lack a specific function that could be used to ascertain proper folding, we instead assessed folding on a basic level by studying their membrane topology and by comparing it to well-established structural models of GPCRs. The topology of the fragments was determined using a reporter assay based on C-terminal green fluorescent protein- or alkaline phosphatase-fusions. N-terminal fusions to Lep or Mistic were used if a periplasmic orientation of the N-terminus of the fragments was expected based on predictions. Fragments fused to Mistic expressed at comparably high levels, whereas Lep fusions were produced to a much lower extent. Though none of the fragments exclusively adopted one orientation, often the correct topology predominated. In addition, systematic analysis of the fragment series suggested that the C-terminal half of the Y4 receptor is more important for adopting the correct topology than the N-terminal part. Using the detergent dodecylphosphocholine, selected fragments were solubilized from the membrane and proved sufficiently stable to allow purification. Finally, as a first step toward reconstituting a functional receptor from two fragments, we observed a physical interaction between complementing fragments pairs upon co-expression.

Bicistronic mRNAs to Enhance Membrane Protein Overexpression

Functional overexpression of membrane proteins is essential for their structural and functional characterization. However, functional overexpression is often difficult to achieve, and frequently either no expression or expression as misfolded aggregates is observed. We present an approach for improving the functional overexpression of membrane proteins in Escherichia coli using transcriptional fusions. The method involves the use of a small additional RNA sequence upstream to the RNA sequence of the target membrane protein and results in the production of a bicistronic mRNA. In contrast to the common approach of translational fusions to enhance protein expression, transcriptional fusions do not require protease treatment and subsequent removal of the fusion protein. Using this strategy, we observed improvements in the quantity and/or the quality of the produced material for several membrane proteins to levels compatible with structural studies. Our analysis revealed that translation of the upstream RNA sequence was not essential for increased expression. Rather, the sequence itself had a large impact on protein yields, suggesting that alternative folding of the transcript was responsible for the observed effect.

Ten Simple Rules for Finishing Your PhD

After years of research and with completion in sight, the final year of the PhD often represents the most challenging time of a students career, in which the ultimate reward is the PhD honor itself. A large investment in time, energy, and motivation is needed, with many tasks to be completed; concluding experiments must be carried out, results interpreted, and a research story mapped out in preparation for writing the final thesis. All the while, administrative obligations need attention (e.g., university credits and mandatory documents), papers may need to be published, students mentored, and due consideration paid to planning for the next career move. Without some form of strategic action plan and the employment of project management skills, students run the risk of becoming overwhelmed and run down or of not meeting their final deadlines. Personal time management and stress resilience are competences that can be developed and honed during this final period of the PhD. Here, we present ten simple rules on how to deal with time issues and conflict situations when facing the last year of a PhD in science. The rules focus on defining research goals in advance and designing a plan of action. Moreover, we discuss the importance of managing relationships with supervisors and colleagues, as well as early career planning.

Biosynthesis and spectroscopic characterization of 2-TM fragments encompassing the sequence of a human GPCR, the Y4 receptor.

This paper presents a divide‐and‐conquer approach towards obtaining solution structures of G protein‐coupled receptors. The human Y4 receptor was dissected into two to three transmembrane helix fragments, which were individually studied by solution NMR. We systematically compared various biosynthetic routes for the expression of the fragments in Escherichia coli and discuss purification strategies. In particular, we have compared the production of transmembrane (TM) fragments as inclusion bodies by using the ΔTrp leader sequence, with membrane‐directed expression by using Mistic as the fusion partner, and developed methods for enzymatic cleavage. In addition, direct expression of two‐TM fragments into inclusion bodies is a successful route in some cases. With the exception of TM13, we could produce all fragments in isotope‐labeled form in quantities sufficient for NMR studies. Almost complete backbone resonance assignment was obtained for the first two helices, as well as for helices 5 and 7, and a high degree was obtained for TM6, while conformational exchange processes resulted in the disappearance of many signals from TM4. In addition, complete assignments were obtained for all residues of the N‐terminal domain, as well as the extracellular and cytosolic loops (with the exception of an undecapeptide segment in the second extracellular loop, EC2) and for the complete cytosolic C‐terminal tail. In total, backbone resonances of 78 % of all residues were assigned for the Y4 receptor. Predictions of secondary structure based on backbone chemical shifts indicate that most residues from the TM regions adopt helical conformations, with exception of those around polar residues or prolines. However, the domain boundaries differ slightly from those predicted for homology models. We suggest that the obtained chemical shifts might be useful in assigning the full‐length receptor.

Mistic's membrane association and its assistance in overexpression of a human GPCR are independent processes

The interaction of the Bacillus subtilis protein Mistic with the bacterial membrane and its role in promoting the overexpression of other membrane proteins are still matters of debate. In this study, we aimed to determine whether individual helical fragments of Mistic are sufficient for its interaction with membranes in vivo and in vitro. To this end, fragments encompassing each of Mistics helical segments and combinations of them were produced as GFP‐fusions, and their cellular localization was studied in Escherichia coli. Furthermore, peptides corresponding to the four helical fragments were synthesized by solid‐phase peptide synthesis, and their ability to acquire secondary structure in a variety of lipids and detergents was studied by circular dichroism spectroscopy. Both types of experiments demonstrate that the third helical fragment of Mistic interacts only with LDAO micelles but does not partition into lipid bilayers. Interestingly, the other three helices interact with membranes in vivo and in vitro. Nevertheless, all of these short sequences can replace full‐length Mistic as N‐terminal fusions to achieve overexpression of a human G‐protein‐coupled receptor in E. coli, although with different effects on quantity and quality of the protein produced. A bioinformatic analysis of the Mistic family expanded the number of homologs from 4 to 20, including proteins outside the genus Bacillus. This information allowed us to discover a highly conserved Shine‐Dalgarno sequence in the operon mstX‐yugO that is important for downstream translation of the potassium ion channel yugO.

How storytelling can help scientists to write better abstracts

Dear Editor, Being a naturally curious storyteller, I learned, can simplify the process of writing successful grant proposals. I am currently a postdoc at the University of Zurich and I have recently applied for a very competitive grant that, if successful, will provide me with the resources to start my own little laboratory. But whilst waiting for the outcome, I wanted to help local PhD students write competitive applications for postdoctoral fellowships. The ability to obtain a postdoctoral fellowship is important for early career scientists. It demonstrates their capacity to secure funding, and increases their chances of joining high-profile laboratories. With a current application success rate of around 10%, postdoctoral fellowships are extremely competitive. Thus, I started a pilot project with the Molecular Life Sciences PhD Program at the University of Zurich with the goal of helping talented young researchers overcome these unfavourable odds. As part of this project, I am now teaching a writing course every Friday afternoon that also includes oneto-one mentoring. Many graduate students have the expectation that being first author on a Nature, Science or Cell paper, combined with a postdoctoral research post in a famous host laboratory, will compensate for a poorly written proposal. But believe me when I tell you, it will not! Therefore, it is never too early to start developing the writing skills that will enable you to produce successful and easy-to-read proposals. In an era of increasing ‘reading noise’, grant reviewers are looking for

Understanding GPCR recognition and folding from NMR studies of fragments

Cotranslational protein folding is a vectorial process, and for membrane proteins, N-terminal helical segments are the first that become available for membrane insertion. While structures of many G-protein coupled receptors (GPCRs) in various states have been determined, the details of their folding pathways are largely unknown. The seven transmembrane (TM) helices of GPCRs often contain polar residues within the hydrophobic core, and some of the helices in isolation are predicted to be only marginally stable in a membrane environment. Here we review our efforts to describe how marginally hydrophobic TM helices of GPCRs integrate into the membrane in absence of all compensating interhelical contacts, ideally capturing early biogenesis events. To this end, we use truncated GPCRs, here referred to as fragments. We present data from the human Y4 and the yeast Ste2p receptors in detergent micelles derived from solution NMR techniques. We find that secondary structure in the fragments is similar to corresponding parts of the entire receptors. However, uncompensated polar or charged residues destabilize the helices, and prevent proper integration into the lipid bilayer, in agreement with the biophysical scales from Wimley and White for the partitioning of amino acids into the membrane-interior. We observe that the stability and integration of single TM helices is improved by adding neighboring helices. We describe a topology study, in which all possible forms of the Y4 receptor were made so that the entire receptor is truncated from the N-terminus by one TM helix at a time. We discover that proteins with an increasing number of helices assume a more defined topology. In a parallel study, we focused on the role of extracellular loops in ligand recognition. We demonstrate that transferring all loops of the human Y1 receptor onto the E. coli outer membrane protein OmpA in a suitable topology results in a chimeric receptor that displays, albeit reduced, affinity and specificity for the cognate ligand. Our data indicate that not all TM helices will spontaneously insert into the helix, and we suggest that at least for some GPCRs, N-terminal segments might remain associated with the translocon until their interacting partners are biosynthesized.

Efficient Screening and Optimization of Membrane Protein Production in Escherichia coli

Escherichia coli is one of the most widely used expression hosts for membrane proteins. However, establishing conditions for its recombinant production of membrane proteins remains difficult. Attempts to produce membrane proteins frequently result in either no expression or expression as misfolded aggregates. We developed an efficient pipeline for improving membrane protein overexpression in E. coli that is based on two approaches. The first involves transcriptional fusions, small additional RNA sequences upstream of the target open reading frame, to overcome no or poor overall expression levels. The other is based on a tunable promoter in combination with a fusion to green fluorescent protein serving as a reporter for the folding state of the target membrane protein. The latter combination allows adjusting the membrane protein expression rate to the downstream folding capacity, in order to decrease the formation of protein aggregates. This pipeline has proven successful for the efficient and parallel optimization of a diverse set of membrane proteins.

Getting ready to postdoc

g s Many PhD students have aspirations of pursuing an academic career [1] and so need to apply for postdoctoral training as their first career step. The ‘ideal’ postdoc should be carried out abroad, with the aim of publishing firstauthor papers in high-impact journals, developing a research niche to foster future grant applications, whilst cultivating a solid collaborative network (Science career blog: Choosing your postdoc position, http://www. science mag.org/careers/2015/06/cho osing-your-postdoc-position). But becoming a group leader requires enormous sacrifices on many levels, including a commitment to do the science sometimes at the expense of a favorable work-life balance, a willingness to relocate, the ability to publish and secure funding, as well as coping with competition and rejection [2]. For these reasons, it is crucial that PhD students enter this process with welldeveloped self-awareness and a full understanding of the uniquely rigorous requirements of an academic career. Equally important, they should know how to identify a future laboratory to suit their needs and to make successful applications for postdoctoral fellowships. Twenty-five PhD students, enrolled under the umbrella of the Life Science Graduate School of ETH/University of Zurich, attended a workshop on the 1st and 2nd February 2017 at the University of Zurich, to gain insights into transitioning on to do a postdoc. The workshop was delivered by two trainers with a combined expertise in funding