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•Method to follow protein unfolding in real-time with atomic resolution.•Unfolding intermediates are not present in solution at detectable levels.•Protein degradation is tightly ...coupled to unfolding.
Protein quality control systems are essential to maintain a healthy proteome. They often consist of an unfoldase unit, typically an AAA+ ATPase, coupled with a protease unit. In all kingdoms of life, they function to eliminate misfolded proteins, and thus prevent that their aggregates do harm to the cell, and to rapidly regulate protein levels in the presence of environmental changes. Despite the huge progress made in the past two decades in understanding the mechanism of function of protein degradation systems, the fate of the substrate during the unfolding and proteolytic processes remains poorly understood. Here we exploit an NMR-based approach to monitor GFP processing by the archaeal PAN unfoldase and the PAN–20S degradation system in real time. We find that PAN-dependent unfolding of GFP does not involve the release of partially-folded GFP molecules resulting from futile unfolding attempts. In contrast, once stably engaged with PAN, GFP molecules are efficiently transferred to the proteolytic chamber of the 20S subunit, despite the only weak affinity of PAN for the 20S subunit in the absence of substrate. This is essential to guarantee that unfolded but not proteolyzed proteins are not released into solution, where they would form toxic aggregates. The results of our studies are in good agreement with previous results derived from real-time small-angle-neutron-scattering experiments and have the advantage of allowing the investigation of substrates and products at amino-acid resolution.
Recombinant protein expression systems that produce high yields of pure proteins and multi‐protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists ...using X‐ray crystallography and cryo‐electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion‐tag to generate recombinant proteins using suspension‐cultured HEK293F cells. YFP is a dual‐function tag that enables direct visualization and fluorescence‐based selection of high expressing clones for and rapid purification using a high‐stringency, high‐affinity anti‐GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression.
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•An ITO (Indium Titanium oxide) based GFP (Green Fluorescent Protein) immobilized electrode (GFP/ITO) was developed to study GFP photobleaching electrochemically.•Electrochemical ...Impedance Spectroscopy (EIS) was used to study changes in impedance of immobilized GFP and correlated with decrease in its fluorescence intensity.•Similar studies were performed with Streptavidin-TMR (tetramethylrhodamine) and Bovine Serum Albumin (BSA) immobilized to ITO for comparative analysis.•It is one of the first kind of study to detect fluorescence changes in GFP using an electrochemical approach.
Electron transport and transition within a molecule are involved in some of the major cellular and biochemical processes in an organism. In proteins, electron transfer is generally characterized by two mechanisms, electron tunnelling and electron hopping. Green Fluorescent Protein (GFP) is the most widely used fluorescent protein in various biological studies. We know that folding occurs in GFP followed by an autocatalytic cyclization and finally oxidation to form a chromophore. The fluorescence phenomenon would cease in proteins which have lost their native structure. In this paper, we have immobilized Strep-TMR (Streptavidin-TMR), BSA (Bovine Serum Albumin) and GFP on a solid surface and checked its functionality under a fluorescence microscope. We also measured the impedance of these bound proteins and found out that ωmax of the Nyquist plot in GFP was less than that of Strep-TMR and BSA. However, when GFP was photobleached by light exposure at various time intervals, an increase in ωmax was observed. This photobleaching and impedimetric correlation observed in case of GFP was compared with the other two proteins as well. Studies have suggested that a number of mechanisms are involved in photobleaching, like structural changes due to photodegradation, ceasing of electron transport or the completion of absorption emission cycles of a fluorophore. In our study, it seems that the electrons responsible for fluorescence in GFP might be responsible for the changes in the ωmax of the Nyquist plot of EIS system. This study is one of the first few approaches that has tried to establish a relationship between fluorescence and electrochemical changes within a fluorescent protein upon photoexposure.
Plasmolysis is a typical response of plant cells exposed to hyperosmotic stress. The loss of turgor causes the violent detachment of the living protoplast from the cell wall. The plasmolytic process ...is mainly driven by the vacuole. Plasmolysis is reversible (deplasmolysis) and characteristic to living plant cells. Obviously, dramatic structural changes are required to fulfill a plasmolytic cycle. In the present paper, the fate of cortical microtubules and actin microfilaments is documented throughout a plasmolytic cycle in living cells of green fluorescent protein (GFP) tagged Arabidopsis lines. While the microtubules became wavy and highly bundled during plasmolysis, cortical filamentous actin remained in close vicinity to the plasma membrane lining the sites of concave plasmolysis and adjusting readily to the diminished size of the protoplast. During deplasmolysis, cortical microtubule re-organization progressed slowly and required up to 24 h to complete the restoration of the original pre-plasmolytic pattern. Actin microfilaments, again, recovered faster and organelle movement remained intact throughout the whole process. In summary, the hydrostatic skeleton resulting from the osmotic state of the plant vacuole "overrules" the stabilization by cortical cytoskeletal elements.
Highlights • New production platforms for VHH and VHH-Fc antibodies were recently explored. • VHHs have distinct properties and often outperform conventional antibodies. • Several VHHs for protein ...purification and localisation are commercially available.
Satellite cells are myogenic progenitors that reside on the myofiber surface and support skeletal muscle repair. We used mice in which satellite cells were detected by GFP expression driven by nestin ...gene regulatory elements to define age-related changes in both numbers of satellite cells that occupy hindlimb myofibers and their individual performance. We demonstrate a reduction in satellite cells per myofiber with age that is more prominent in females compared to males. Satellite cell loss also persists with age in myostatin-null mice regardless of increased muscle mass. Immunofluorescent analysis of isolated myofibers from nestin–GFP/Myf5nLacZ/+ mice reveals a decline with age in the number of satellite cells that express detectable levels of βgal. Nestin–GFP expression typically diminishes in primary cultures of satellite cells as myogenic progeny proliferate and differentiate, but GFP subsequently reappears in the Pax7+ reserve population. Clonal analysis of sorted GFP+ satellite cells from hindlimb muscles shows heterogeneity in the extent of cell density and myotube formation among colonies. Reserve cells emerge primarily within high-density colonies, and the number of clones that produce reserve cells is reduced with age. Thus, satellite cell depletion with age could be attributed to a reduced capacity to generate a reserve population.
► Purification and characterization of Clp proteins from Bradyrhizobium japonicum. ► ClpA of B. japonicum is autodegraded in a ClpAP-dependent manner. ► FixK2, a key regulatory protein, is ...specifically degraded by ClpAP1. ► The C-terminus of FixK2 is the main recognition site for ClpA.
FixK2 is a CRP-like transcription factor that controls the endosymbiotic lifestyle of Bradyrhizobium japonicum. The reason for its noticeable protease sensitivity was explored here. The repertoire of Clp chaperone-proteases in B. japonicum was examined, and specifically ClpAP1 and ClpXP1 were purified and tested. FixK2 was found to be degraded by ClpAP1 but not by ClpXP1. Degradation was inhibited by the ClpS1 adaptor protein, indicating that FixK2 is a direct substrate for ClpAP1. The last 12 amino acids of FixK2 appeared to be recognized by ClpA. The results suggest that the ClpAP system is involved in the cellular turnover of FixK2.
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G ...protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
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•PKA RIα undergoes liquid-liquid phase separation in response to cAMP dynamics•RIα condensates dynamically sequester cAMP and retain high PKA activity•RIα phase separation is necessary for effective cAMP compartmentation•A PKA oncoprotein disrupts RIα bodies, leading to aberrant signaling and growth
cAMP-responsive condensate formation by PKA’s RIα subunit controls local signaling, and disruption of phase separation in this context contributes to tumorigenesis.
Despite the partial disorder‐to‐order transition that intrinsically disordered proteins often undergo upon binding to their partners, a considerable amount of residual disorder may be retained in the ...bound form, resulting in a fuzzy complex. Fuzzy regions flanking molecular recognition elements may enable partner fishing through non‐specific, transient contacts, thereby facilitating binding, but may also disfavor binding through various mechanisms. So far, few computational or experimental studies have addressed the effect of fuzzy appendages on partner recognition by intrinsically disordered proteins. In order to shed light onto this issue, we used the interaction between the intrinsically disordered C‐terminal domain of the measles virus (MeV) nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein as model system. After binding to XD, the N‐terminal region of NTAIL remains conspicuously disordered, with α‐helical folding taking place only within a short molecular recognition element. To study the effect of the N‐terminal fuzzy region on NTAIL/XD binding, we generated N‐terminal truncation variants of NTAIL, and assessed their binding abilities towards XD. The results revealed that binding increases with shortening of the N‐terminal fuzzy region, with this also being observed with hsp70 (another MeV NTAIL binding partner), and for the homologous NTAIL/XD pairs from the Nipah and Hendra viruses. Finally, similar results were obtained when the MeV NTAIL fuzzy region was replaced with a highly dissimilar artificial disordered sequence, supporting a sequence‐independent inhibitory effect of the fuzzy region.
A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a ...native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.
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