Transition metal ion FRET between a noncanonical fluorescent amino acid incorporated into TRPV1 and metal ions bound to the cell plasma can be used to measure distances and dynamics between cytosolic domains of proteins and the membrane.
The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+. Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.
big rectangle). This nuclear plasticity, measured as projected nuclear area fluctuations, showed a non-monotonous relation to actin polymerization state. Also, myosin contractility was determined to be necessary for such nucleus plasticity. The effect of cytoskeletal organization and their active forces on chromatin plasticity was further quantified by tracking the dynamics of condensed chromatin regions, which showed increased dynamics corresponding to enhanced nuclear plasticity. In summary, using cells of defined geometries to specify cytoskeletal organization, our work demonstrates the role of active cytoskeletal forces in regulating nuclear and chromatin plasticity. The prebiotic formation of biopolymers (specifically DNA, RNA, and proteins) has long been a mystery and is important for understanding the origin of life on earth. These bio-molecules are composed of building blocks that would have been dispersed in early oceans. Our previous work has shown that RNA bases and ribose bind to and stabilize fatty acid vesicles [Black et al. PNAS 110, 13272 (2013)]. Our results implied that the building blocks of a biological polymer could have spontaneously associated with components of the first membranes to form stable structures. We have now shown that protein building blocks, too, stabilize fatty acid vesicles against salt-induced flocculation. Using spectrophotometry, we measured the presence of flocs (and other structures) in fatty acid solutions, with and without amino acids and over a range of temperatures. Using fluorescence microscopy, we identified the structures that caused changes in absorbance in our spectrophotometric assays. We found that the two most hydrophobic prebiotic amino acids, leucine and isoleucine, prevent salt-induced flocculation. Moreover, although alanine and glycine, which are less hydrophobic, had little effect on flocculation, dipeptides composed of these amino acids preserved vesicles in the presence of salt even at 60 degrees C. These vesicles appeared to be primarily multilamellar structures, which may promote reactions between components of biopolymers more effectively than unilamellar vesicles. Thus prebiotic membranes could have facilitated the formation of peptides by bringing amino acids together, and peptides could have increased the formation of stable membranes. Such an auto-amplifying system, combined with selection for more effective peptides, could have led to the first cells. 2746-Pos Board B176Measurement of the Viscosity of E. coli Membranes using Molecular Rotors and Flim
Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co 2+ bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.
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