Biology subjects

Tamm, L. K.

Publications and source records attributed to Tamm, L. K..

4 recordsLinked to original sources

bioRxiv2022

Serinc5 restricts HIV membrane fusion by altering lipid order and heterogeneity in the viral membrane

The host restriction factor, Serinc5, incorporates into budding HIV particles and inhibits their infection by an incompletely understood mechanism. We have previously reported that Serinc5 but not its paralogue, Serinc2, blocks HIV cell entry by membrane fusion, specifically by inhibiting fusion pore formation and dilation. A compelling body of work also suggests Serinc5 may alter the conformation and clustering of the HIV fusion protein, Env. To contribute an additional perspective to the developing model of Serinc5 restriction, we assessed Serinc2 and Serinc5s effects on HIV pseudoviral membranes. Using fluorescence lifetime imaging with an order sensitive dye, FLIPPER-TR, and by measuring pseudoviral membrane thickness via cryo electron microscopy (cryoEM), Serinc5 was found to increase membrane heterogeneity, skewing the distribution towards a larger fraction of the viral membrane in an ordered phase. We also directly observed for the first time the coexistence of membrane domains within individual viral membrane envelopes. Using a TIRF-based single particle fusion assay, we found that incorporation of exogenous phosphatidylethanolamine (PE) into the viral membrane rescued HIV pseudovirus fusion from restriction by Serinc5, which was accompanied by decreased membrane heterogeneity and order. This effect was specific for PE and did not depend on acyl chain length or saturation. Together, these data suggest that Serinc5 alters multiple interrelated properties of the viral membrane--lipid chain order, rigidity, line tension, and lateral pressure--which decrease accessibility of fusion intermediates and disfavor completion of fusion. These biophysical insights into Serinc5 restriction of HIV infectivity could contribute to the development of novel antivirals that exploit the same weaknesses of HIV and potentially other enveloped viruses.

biophysics↗

bioRxiv2020

De novo design of transmembrane beta-barrels

The ability of naturally occurring transmembrane {beta}-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a "hypothesis, design and test" approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the {beta}-turns and {beta}-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history. One sentence summarySuccess in de novo design of transmembrane {beta}-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

synthetic biology↗

bioRxiv2020

Conserved Arginine Residues in Synaptotagmin 1 Regulate Fusion Pore Expansion Through Membrane Contact

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the probably and kinetics of fusion but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a novel model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

neuroscience↗

bioRxiv2019

Synaptotagmin-7 endows a population of chromaffin cell vesicles with enhanced calcium sensing and delayed content release properties

Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite its undoubted importance, questions remain as to the functional and physiological role of Syt-7 in secretion. We examined this issue using two distinct preparations - mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived vesicles expressing either Syt-7 or Syt-1. First, we report using immunofluorescence that Syt-7 exhibits a punctate intracellular distribution consistent with its sorting to organelles, including dense core vesicles. We also find that the likelihood of vesicle fusion in KO cells is markedly lower than in WT cells. When fusion does occur, cargoes are discharged more rapidly when only Syt-1 is available to facilitate release. A distinctive characteristic of KO cells is that secretion runs down after prolonged cholinergic stimulation. In contrast, exocytosis persists in WT cells even with extended exposure to acetylcholine, suggesting a key role for Syt-7 in sustaining the secretory response. To determine the extent to which the aforementioned results are attributable purely to Syt-7, vesicles expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to vesicle fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.

biophysics↗