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Cotranslational folding of small protein domains within the ribosome exit tunnel may be an important cellular strategy to avoid protein misfolding. However, the pathway of cotranslational folding has so far been described only for a few proteins, and therefore, it is unclear whether folding in the ribosome exit tunnel is a common feature for small protein domains. Here, we have analyzed nine small protein domains and determined at which point during translation their folding generates sufficient force on the nascent chain to release translational arrest by the SecM arrest peptide, both in vitro and in live E. coli cells. We find that all nine protein domains initiate folding while still located well within the ribosome exit tunnel. [ABSTRACT FROM AUTHOR]

The article discusses a two-day workshop based on postdoctoral studies for PhD students, at the University of Zurich, on 1 and 2 February, 2017. It describes how the students were taken through a process of analysing potential academic paths on the first day. It also describes how the students were taken through the process of writing scientific narratives on the second day, using filmmaker Randy Olson's methods, as well as an oral exam.

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. [ABSTRACT FROM AUTHOR]

The author discusses ten rules that help finish the Doctor of Philosophy (PhD) degree and highlights importance of managing relationships with colleagues and supervisors. It suggests preparing plan of action for last year of PhD in advance, selecting activities that will be included in the plan of action, and finalizing experiments and writing the thesis.

Abstract: 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. [Copyright &y& Elsevier]

[Display omitted] • This analysis sheds light on the dynamical nature of GPCR-G-protein complexes. • The receptor' C-terminal tail and the alpha-helical domain of G-alpha are dynamic. • The movements observed are similar for other published GPCR complexes. • This analysis can guide the selection of Fabs to limit conformational heterogeneity G-protein coupled receptors (GPCRs) are among the most versatile signal transducers in the cell. Once activated, GPCRs sample a large conformational space and couple to G-proteins to initiate distinct signaling pathways. The dynamical behavior of GPCR-G-protein complexes is difficult characterize structurally, and it might hinder obtaining routine high-resolution density maps in single-particle reconstructions. Here, we used variability analysis on the rhodopsin-G i -Fab16 complex cryo-EM dataset, and the results provide insights into the dynamic nature of the receptor-complex interaction. We compare the outcome of this analysis with recent results obtained on the cannabinoid-G i - and secretin-G s -receptor complexes. Despite differences related to the biochemical compositions of the three samples, a set of consensus movements emerges. We anticipate that systematic variability analysis on GPCR-G-protein complexes may provide useful information not only at the biological level, but also for improving the preparation of more stable samples for cryo-EM single-particle analysis. [ABSTRACT FROM AUTHOR]

Cyclic nucleotide-gated (CNG) channels play a central role in rod and cone photoreceptors of the vertebrate retina. In photoreceptors, light triggers a series of biochemical reactions that ultimately close CNG channels and evoke a brief voltage pulse, a signal that is later passed on to the brain. Malfunction of CNG channels can lead to loss of vision. Thus, understanding their function in atomic and mechanistic detail is important. Because of the complex subunit stoichiometry of these channels, elucidation of their structure has proved challenging. Recently, several cryoelectron microscopy (EM) structures of rod and cone CNG channels revealed unexpected structural features. We compare these structures side by side and highlight similarities and differences in key structural elements. We discuss the implications of the channels' structure for questions about their gating, ion permeation, and modulation. These results inform new strategies to further characterize the structural basis of CNG channels functioning in rods and cones. Cyclic nucleotide-gated (CNG) channels in rod and cone photoreceptors play a central role in vision. Mutations in CNG channels lead to visual impairment or even blindness. The development of drugs to treat conditions involving CNG channel dysfunction has been slow, partly due to the lack of atomic structures of these channels. Comparison of the atomic structures of CNG channels from rod and cone photoreceptors in closed and open states reveals key elements that control gating and ion permeation. There are characteristic differences between homomeric CNGA and heteromeric CNGA/CNGB channels. The rod CNG channel offers first insights into the opening of the asymmetric ion conduction pathway. An arginine residue in the pore of the CNGB subunit creates a second gate in the channel pore. The residue forming the CNGB gate in cones varies with species. [ABSTRACT FROM AUTHOR]

The authors reflect on some rules for obtaining assistance from online scientific communities with the use of different communication tools. The authors the said tools allows a scientist to contact people skilled quickly in a specialized field, however correct online scientific community must be known to solve difficult problems. They say that to get a good answer, people must state the question distinctly in a concise way, and must also learn to make customs before posting queries.

The article presents the author's views on the use of storytelling by scientists to help them write abstracts for grant proposals, and mentions that the author has applied for a competitive grant. Topics discussed include the book "Houston, We Have a Narrative: Why Science Needs Story" written by filmmaker Randy Olson focusing on storytelling and explaining the use of communication in science; and the book "Four Steps to Funding" written by Morgan Giddings.

The article discusses the advantages and disadvantages for scientists that go abroad to do research. Topics include the author's experience of leaving the University of Parma in Italy to work at the University of Zurich (UZH) in Switzerland, where he is pursing a postdoctoral degree (Ph.D.), his move to study membrane proteins at scientist Gunnar von Heijne's laboratory at Sweden's Stockholm University, and the personal and cultural growth opportunities of travel and relocation.

Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available. [ABSTRACT FROM AUTHOR]

Recent work has demonstrated that cotranslational folding of proteins or protein domains in, or in the immediate vicinity of, the ribosome exit tunnel generates a pulling force on the nascent polypeptide chain that can be detected using a so-called translational arrest peptide ( AP) engineered into the nascent chain as a force sensor. Here, we show that AP-based force measurements combined with systematic Ala and Trp scans of a zinc-finger domain that folds in the exit tunnel can be used to identify the residues that are critical for intraribosomal folding. Our results suggest a general approach to characterize the folded state(s) that may form as a protein domain moves progressively down the ribosome exit tunnel. [ABSTRACT FROM AUTHOR]

[Display omitted] • The cryo-EM density map of the human CNG CNGA1/CNGB1 channel reveals the presence of an additional density. • This additional density fits well the structure of calmodulin. • Calmodulin plays a fundamental role modulating of the activity of native CNG channels, however the structural basis for this modulation remain unclear. • We propose that the binding of calmodulin to the CNG channel explains the partially open state reported by Xue et al. (Neuron 110, 1-10, 2021). • Two models are provided to interpret the cryo-EM density map of the human CNG CNGA1/CNGB1 channel. The recently reported structure of the human CNGA1/CNGB1 CNG channel in the open state (Xue et al., 2021a) shows that one CNGA1 and one CNGB1 subunit do not open the central hydrophobic gate completely upon cGMP binding. This is different from what has been reported for CNGA homomeric channels (Xue et al., 2021b; Zheng et al., 2020). In seeking to understand how this difference is due to the presence of the CNGB1 subunit, we find that the deposited density map (Xue et al., 2021a) (EMDB 24465) contains an additional density not reported in the images of the original publication. This additional density fits well the structure of calmodulin (CaM), and it unambiguously connects the newly identified D-helix of CNGB1 to one of the CNGA1 helices (A1 R) participating in the coiled-coil region. Interestingly, the CNGA1 subunit that engages in the interaction with this additional density is the one that, together with CNGB1, does not open completely the central gate. The sequence of the D-helix of CNGB1 contains a known CaM-binding site of exquisitely high affinity – named CaM2 (Weitz et al., 1998) –, and thus the presence of CaM in that region is not surprising. The mechanism through which CaM reduces currents across the membrane by acting on the native channel (Bauer, 1996; Hsu and Molday, 1993; Weitz et al., 1998) remains unclear. We suggest that the presence of CaM may explain the partially open central gate reported by Xue et al. (2021a). The structure of the open and closed states of the CNGA1/CNGB1 channel may be different with and without CaM present. [ABSTRACT FROM AUTHOR]