scholarly journals Dynamic membrane topology in an unassembled membrane protein

2019 ◽  
Author(s):  
Maximilian Seurig ◽  
Moira Ek ◽  
Gunnar von Heijne ◽  
Nir Fluman
Keyword(s):  
Membrane Protein ◽  
Transmembrane Helix ◽  
Membrane Topology ◽  
Topological Dynamics ◽  
Membrane Insertion ◽  
Dynamic Membrane ◽  
Membrane Protein Topology ◽  
Small Multidrug Resistance ◽  
Kinetics Of

AbstractHelical membrane proteins constitute roughly a quarter of all proteomes and perform diverse biological functions. To avoid aggregation, they undergo cotranslational membrane insertion and are typically assumed to attain stable transmembrane topologies immediately upon insertion. To what extent post-translational changes in topology are possible in-vivo and how they may affect biogenesis is incompletely understood. Here, we show that monomeric forms of Small Multidrug Resistance (SMR) proteins display topological dynamics, where the N-terminal transmembrane helix equilibrates between membrane-inserted and non-inserted states. We characterize the kinetics of the process and show how the composition of the helix regulates the topological dynamics. We further show that topological dynamics is a property of the unassembled monomeric protein, as the N-terminal helix becomes fixed in a transmembrane disposition upon dimerization. Membrane protein topology can thus remain dynamic long after cotranslational membrane insertion, and can be regulated by later assembly processes.

scholarly journals Insights into the key determinants of membrane protein topology enable the identification of new monotopic folds

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sonya Entova ◽  
Jean-Marc Billod ◽  
Jean-Marie Swiecicki ◽  
Sonsoles Martín-Santamaría ◽  
Barbara Imperiali
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayer ◽  
Membrane Topology ◽  
Membrane Protein Topology ◽  
New Information ◽  
Single Face ◽  
Membrane Spanning ◽  
Definition Of ◽  
Computational Analyses

Monotopic membrane proteins integrate into the lipid bilayer via reentrant hydrophobic domains that enter and exit on a single face of the membrane. Whereas many membrane-spanning proteins have been structurally characterized and transmembrane topologies can be predicted computationally, relatively little is known about the determinants of membrane topology in monotopic proteins. Recently, we reported the X-ray structure determination of PglC, a full-length monotopic membrane protein with phosphoglycosyl transferase (PGT) activity. The definition of this unique structure has prompted in vivo, biochemical, and computational analyses to understand and define key motifs that contribute to the membrane topology and to provide insight into the dynamics of the enzyme in a lipid bilayer environment. Using the new information gained from studies on the PGT superfamily we demonstrate that two motifs exemplify principles of topology determination that can be applied to the identification of reentrant domains among diverse monotopic proteins of interest.

scholarly journals Intra-helical salt bridge contribution to membrane protein insertion

2021 ◽  
Author(s):  
Gerard Duart ◽  
John Lamb ◽  
Arne Elofsson ◽  
Ismael Mingarro
Keyword(s):  
Amino Acids ◽  
Membrane Protein ◽  
Electrostatic Interactions ◽  
Salt Bridge ◽  
Protein Stabilization ◽  
Membrane Insertion ◽  
Salt Bridges ◽  
Protein Insertion

ABSTRACTSalt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an important role in protein stabilization. This has a more prevalent effect in membrane proteins where polar amino acids are exposed to a very hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. This can sometimes be avoided by TM region rearrangements after insertion, but it is also possible that the formation of salt bridges could decrease the cost of membrane integration. However, the presence of intra-helical salt bridges in TM domains and their effect on insertion has not been properly studied yet. In this work, we use an analytical pipeline to study the prevalence of charged pairs of amino acid residues in TM α-helices, which shows that potentially salt-bridge forming pairs are statistically over-represented. We then selected some candidates to experimentally determine the contribution of these electrostatic interactions to the translocon-assisted membrane insertion process. Using both in vitro and in vivo systems, we confirm the presence of intra-helical salt bridges in TM segments during biogenesis and determined that they contribute between 0.5-0.7 kcal/mol to the apparent free energy of membrane insertion (ΔGapp). Our observations suggest that salt bridge interactions can be stabilized during translocon-mediated insertion and thus could be relevant to consider for the future development of membrane protein prediction software.

scholarly journals Residue-by-residue analysis of cotranslational membrane protein integration in vivo

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Felix Nicolaus ◽  
Ane Metola ◽  
Daphne Mermans ◽  
Amanda Liljenström ◽  
Ajda Krč ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Profile Analysis ◽  
Residue Analysis ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Dependent Manner ◽  
Integration Process ◽  
Pulling Forces ◽  
Exit Tunnel

We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the ‘sliding’ model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon.

scholarly journals Analysis of F Factor TraD Membrane Topology by Use of Gene Fusions and Trypsin-Sensitive Insertions

1999 ◽  
Vol 181 (19) ◽  
pp. 6108-6113 ◽  
Author(s):  
Martin H. Lee ◽  
Nick Kosuk ◽  
Jeannie Bailey ◽  
Beth Traxler ◽  
Colin Manoil
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Subcellular Location ◽  
Membrane Topology ◽  
Gene Fusions ◽  
Reporter Protein ◽  
Membrane Protein Topology ◽  
F Factor ◽  
Reporter Proteins ◽  
Membrane Spanning

ABSTRACT This report describes a procedure for characterizing membrane protein topology which combines the analysis of reporter protein hybrids and trypsin-sensitive 31-amino-acid insertions generated by using transposons ISphoA/in and ISlacZ/in. Studies of the F factor TraD protein imply that the protein takes on a structure with two membrane-spanning sequences and amino and carboxyl termini facing the cytoplasm. It was possible to assign the subcellular location of one region for which the behavior of fused reporter proteins was ambiguous, based on the trypsin cleavage behavior of a 31-residue insertion.

scholarly journals Stable membrane topologies of small dual-topology membrane proteins

2017 ◽  
Author(s):  
Nir Fluman ◽  
Victor Tobiasson ◽  
Gunnar von Heijne
Keyword(s):  
Membrane Proteins ◽  
Membrane Topology ◽  
Multidrug Resistance Proteins ◽  
Flip Flop ◽  
Biologically Relevant ◽  
Small Multidrug Resistance ◽  
Fixed Orientation ◽  
Static View ◽  
The Individual

AbstractThe topologies of α-helical membrane proteins are generally thought to be determined during their cotranslational insertion into the membrane. It is typically assumed that membrane topologies remain static after this process has ended. Recent findings, however, question this static view by suggesting that some parts of, or even the whole protein, can reorient in the membrane on a biologically relevant time scale. Here, we focus on anti-parallel homo-or hetero-dimeric Small Multidrug Resistance proteins, and examine whether the individual monomers can undergo reversible topological inversion (flip-flop) in the membrane until they are trapped in a fixed orientation by dimerization. By perturbing dimerization using various means, we show that the membrane topology of a monomer is unaffected by the presence or absence of its dimerization partner. Thus, membrane-inserted monomers attain their final topologies independently of dimerization, suggesting that wholesale topological inversion is an unlikely event in vivo.

scholarly journals Dynamic membrane topology in an unassembled membrane protein

2019 ◽  
Vol 15 (10) ◽  
pp. 945-948 ◽  
Author(s):  
Maximilian Seurig ◽  
Moira Ek ◽  
Gunnar von Heijne ◽  
Nir Fluman
Keyword(s):  
Membrane Protein ◽  
Membrane Topology ◽  
Dynamic Membrane

scholarly journals In Vivo Membrane Topology of Escherichia coli SecA ATPase Reveals Extensive Periplasmic Exposure of Multiple Functionally Important Domains Clustering on One Face of SecA

2006 ◽  
Vol 282 (7) ◽  
pp. 4661-4668 ◽  
Author(s):  
Lucia B. Jilaveanu ◽  
Donald B. Oliver
Keyword(s):  
Model Building ◽  
Protein Translocation ◽  
Membrane Topology ◽  
Membrane Insertion ◽  
Conducting Channel ◽  
Domain Specific ◽  
Single Face ◽  
Dependent Protein ◽  
Extensive Collection

The Sec-dependent protein translocation pathway promotes the transport of proteins into or across the bacterial plasma membrane. SecA ATPase has been shown to be a nanomotor that associates with its protein cargo as well as the SecYEG channel complex and to undergo ATP-driven cycles of membrane insertion and retraction that promote stepwise protein translocation. Previous studies have shown that both the 65-kDa N-domain and 30-kDa C-domain of SecA appear to undergo such membrane cycling. In the present study we performed in vivo sulfhydryl labeling of an extensive collection of monocysteine secA mutants under topologically specific conditions to identify regions of SecA that are accessible to the trans side of the membrane in its membrane-integrated state. Our results show that distinct regions of five of six SecA domains were labeled under these conditions, and such labeling clusters to a single face of the SecA structure. Our results demarcate an extensive face of SecA that interacts with SecYEG and is in fluid contact with the protein-conducting channel. The observed domain-specific labeling patterns should also provide important constraints on model building efforts in this dynamic system.

Membrane proteins: from bench to bits

2011 ◽  
Vol 39 (3) ◽  
pp. 747-750 ◽  
Author(s):  
Gunnar von Heijne
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
X Ray ◽  
Protein Topology ◽  
Helix Bundle ◽  
Transmembrane Segments ◽  
Membrane Protein Topology

Membrane proteins currently receive a lot of attention, in large part thanks to a steady stream of high-resolution X-ray structures. Although the first few structures showed proteins composed of tightly packed bundles of very hydrophobic more or less straight transmembrane α-helices, we now know that helix-bundle membrane proteins can be both highly flexible and contain transmembrane segments that are neither very hydrophobic nor necessarily helical throughout their lengths. This raises questions regarding how membrane proteins are inserted into the membrane and fold in vivo, and also complicates life for bioinformaticians trying to predict membrane protein topology and structure.

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