Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit

Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.

The role of hydrophobic matching on transmembrane helix packing in cells

Folding and packing of membrane proteins are highly influenced by the lipidic component of the membrane. Here, we explore how the hydrophobic mismatch (the difference between the hydrophobic span of a transmembrane protein region and the hydrophobic thickness of the lipid membrane around the protein) influences transmembrane helix packing in a cellular environment. Using a ToxRED assay in Escherichia coli and a Bimolecular Fluorescent Complementation approach in human-derived cells complemented by atomistic molecular dynamics simulations we analyzed the dimerization of Glycophorin A derived transmembrane segments. We concluded that, biological membranes can accommodate transmembrane homo-di…

Methodological approaches for the analysis of transmembrane domain interactions: A systematic review

The study of protein-protein interactions (PPI) has proven fundamental for the understanding of the most relevant cell processes. Any protein domain can participate in PPI, including transmembrane (TM) segments that can establish interactions with other TM domains (TMDs). However, the hydrophobic nature of TMDs and the environment they occupy complicates the study of intramembrane PPI, which demands the use of specific approaches and techniques. In this review, we will explore some of the strategies available to study intramembrane PPI in vitro, in vivo, and, in silico, focusing on those techniques that could be carried out in a standard molecular biology laboratory regarding its previous e…

Understanding membrane protein folding and interfaciality

Despite the bast abundance of membrane proteins encoded in the human genome, the understanding of their biosynthesis and folding is far from the knowledge we have of soluble proteins. In the present thesis the main objective is to increase our knowledge on membrane protein biogenesis and folding, from the first steps of folding within the ribosome to the native structure acquisition in the membrane. Hydrophobicity, helicity and length of the polypeptide chain have been stablished as the major determinants for alpha-helical conformation adoption within the ribosome exit tunnel. Also, a 'biological' interfacial scale for the 20 naturally occurring amino acids were determined due to the develo…

The Role of Hydrophobic Mismatch on Transmembrane Helix Dimerization in Living Cells

Viral Bcl2s' transmembrane domain interact with host Bcl2 proteins to control cellular apoptosis

© The Author(s) 2020.

Conformational clamping by a membrane ligand activates the EphA2 receptor

AbstractThe EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migratio…

SARS-CoV-2 envelope protein topology in eukaryotic membranes

Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Nt lum /Ct cyt ). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and ho…

Controllable membrane remodeling by a modified fragment of the apoptotic protein Bax.

Intrinsic apoptosis is orchestrated by a group of proteins that mediate the coordinated disruption of mitochondrial membranes. Bax is a multi-domain protein that, upon activation, disrupts the integrity of the mitochondrial outer membrane by forming pores. We strategically introduced glutamic acids into a short sequence of the Bax protein that constitutively creates membrane pores. The resulting BaxE5 peptide efficiently permeabilizes membranes at acidic pH, showing low permeabilization at neutral pH. Atomic force microscopy (AFM) imaging showed that at acidic pH BaxE5 established several membrane remodeling modalities that progressively disturbed the integrity of the lipid bilayer. The AFM…

Folding and insertion of transmembrane helices at the ER

In eukaryotic cells, the endoplasmic reticulum (ER) is the entry point for newly synthesized proteins that are subsequently distributed to organelles of the endomembrane system. Some of these proteins are completely translocated into the lumen of the ER while others integrate stretches of amino acids into the greasy 30 Å wide interior of the ER membrane bilayer. It is generally accepted that to exist in this non-aqueous environment the majority of membrane integrated amino acids are primarily non-polar/hydrophobic and adopt an α-helical conformation. These stretches are typically around 20 amino acids long and are known as transmembrane (TM) helices. In this review, we will consider how tra…

Transmembrane but not soluble helices fold inside the ribosome tunnel

Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form α-helices before integration. Co-translational folding of nascent chains into an α-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire α-helical structure in the ribosom…