Biology subjects

Nyenhuis, S. B.

Publications and source records attributed to Nyenhuis, S. B..

4 recordsLinked to original sources

bioRxiv2024

The translocation assembly module (TAM) catalyzes the assembly of bacterial outer membrane proteins in vitro

The bacterial translocation assembly module (TAM) contains an outer membrane protein (OMP) (TamA) and an elongated periplasmic protein that is anchored to the inner membrane by a single helix (TamB). TAM has been proposed to play a critical role in the assembly of a small subset of OMPs produced by Proteobacteria based on experiments conducted in vivo using tamA and/or tamB deletion or mutant strains and in vitro using biophysical methods. Recent genetic experiments, however, have strongly suggested that TAM promotes phospholipid homeostasis. To test the idea that TAM catalyzes OMP assembly directly, we examined the function of the purified E. coli complex in vitro after reconstituting it into proteoliposomes. Remarkably, we find that TAM catalyzes the assembly of four model OMPs nearly as well as the {beta}-barrel assembly machinery (BAM), a universal heterooligomer that contains a TamA homolog (BamA) and that catalyzes the assembly of almost all E. coli OMPs. Consistent with previous results, both TamA and TamB are required for significant TAM activity. Our results provide strong evidence that although their peripheral subunits are unrelated, both BAM and TAM function as independent OMP insertases. Furthermore, our study describes a new method to gain insights into TAM function.

microbiology↗

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

Partial metal ion saturation of C2 domains primes Syt1-membrane interactions

Synaptotagmin 1 (Syt1) is an integral membrane protein that acts as a Ca2+ sensor of neurotransmitter release. How the Ca2+-sensing function of Syt1 is coupled to its interactions with anionic membranes and synaptic fusion machinery is not well understood. Here, we investigated the dynamics and membrane-binding properties of Syt1 under conditions where its highest affinity Ca2+ sites, which are thought to drive the initial membrane recruitment, are selectively populated by divalent metal ions. To create such protein states for the Ca2+-sensing C2 domains of Syt1, we exploited the unique chemistry of Pb2+, a xenobiotic metal ion that is isostructural and isofunctional to Ca2+. NMR experiments revealed that binding of a single metal ion results in the loss of conformational plasticity of the C2 domain loop regions that are involved in both coordinating metal ions and membrane interactions. In the C2A domain, a single metal ion is sufficient to drive its weak association with PtdSer-containing membranes; in C2B, it enhances the interactions with the signaling lipid PtdIns(4,5)P2. In full-length Syt1, both C2 domains associate with PtdSer-containing membranes, with the depth of insertion modulated by the occupancy of the metal ion sites. Our data suggest that Syt1 adopts a shallow membrane-bound state upon initial recruitment of its C2 domains to the membranes. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, \"prime\" Syt1 for binding a full complement of metal ions required for activation of protein function.

biochemistry↗

bioRxiv2019

Phosphatidylinositol 4,5 bisphosphate controls the cis and trans interactions of synaptotagmin 1

Synaptotagmin 1 acts as the Ca2+-sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here we describe measurements on full-length membrane reconstituted synaptotagmin 1 using site-directed spin labeling where we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate (PIP2), even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+-binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS containing bilayers and will bind in a cis configuration to membranes containing PS even if a PIP2 membrane is presented in trans. The data are consistent with a bridging activity for Syt1 where the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

neuroscience↗