Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

Bañó‐Polo, Manuel; Martínez-Gil, Luis; Wallner, Björn; Nieva, José L.; Elofsson, Arne; Mingarro, Ismael

doi:10.1016/j.jmb.2012.12.001

Cited by 32 publications

(32 citation statements)

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“…1D, center). However, insertion of both HRs into the membrane should result in only G1 receiving a glycan, because, as previously demonstrated (33), G2 in these constructs was too close to the membrane to be efficiently glycosylated (Fig. 1D, left).…”

Section: Isolated Tmv Mp Hydrophobic Regions Are Not Inserted Into Bimentioning

confidence: 53%

“…Previous studies have shown that, in some cases, a neighboring TM helix can promote membrane insertion of a poorly hydrophobic TM region (30)(31)(32)(33). Therefore, we used the in vitro system to investigate the insertion of the two HRs connected by their native loop (residues 61 to 167, shown in gray) (Fig.…”

Section: Isolated Tmv Mp Hydrophobic Regions Are Not Inserted Into Bimentioning

confidence: 99%

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The Tobacco mosaic virus Movement Protein Associates with but Does Not Integrate into Biological Membranes

Peiró

Martínez-Gil

Tamborero

et al. 2014

J Virol

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Plant positive-strand RNA viruses require association with plant cell endomembranes for viral translation and replication, as well as for intra-and intercellular movement of the viral progeny. The membrane association and RNA binding of the Tobacco mosaic virus (TMV) movement protein (MP) are vital for orchestrating the macromolecular network required for virus movement. A previously proposed topological model suggests that TMV MP is an integral membrane protein with two putative ␣-helical transmembrane (TM) segments. Here we tested this model using an experimental system that measured the efficiency with which natural polypeptide segments were inserted into the ER membrane under conditions approximating the in vivo situation, as well as in planta. Our results demonstrated that the two hydrophobic regions (HRs) of TMV MP do not span biological membranes. We further found that mutations to alter the hydrophobicity of the first HR modified membrane association and precluded virus movement. We propose a topological model in which the TMV MP HRs intimately associate with the cellular membranes, allowing maximum exposure of the hydrophilic domains of the MP to the cytoplasmic cellular components. IMPORTANCE To facilitate plant viral infection and spread, viruses encode one or more movement proteins (MPs) that interact with ER membranes. The present work investigated the membrane association of the 30K MP of

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Section: Isolated Tmv Mp Hydrophobic Regions Are Not Inserted Into Bimentioning

confidence: 53%

Section: Isolated Tmv Mp Hydrophobic Regions Are Not Inserted Into Bimentioning

confidence: 99%

The Tobacco mosaic virus Movement Protein Associates with but Does Not Integrate into Biological Membranes

Peiró

Martínez-Gil

Tamborero

et al. 2014

J Virol

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“…Because of a lack of good assays, this idea has been difficult to test experimentally. The best support for it thus far has been obtained by end-point studies that compare the efficiency of membrane insertion of marginally hydrophobic transmembrane helices (TMHs) in their natural sequence context and when interacting residues in the protein have been removed by mutagenesis (8)(9)(10)(11)(12)(13), but such studies cannot identify the precise point during the translation-insertion-folding process when interactions between TMHs first develop.…”

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confidence: 99%

Cotranslational folding of membrane proteins probed by arrest-peptide–mediated force measurements

Cymer

Heijne

2013

Proc. Natl. Acad. Sci. U.S.A.

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Polytopic membrane proteins are inserted cotranslationally into target membranes by ribosome-translocon complexes. It is, however, unclear when during the insertion process specific interactions between the transmembrane helices start to form. Here, we use a recently developed in vivo technique to measure pulling forces acting on transmembrane helices during their cotranslational insertion into the inner membrane of Escherichia coli to study the earliest steps of tertiary folding of five polytopic membrane proteins. We find that interactions between residues in a C-terminally located transmembrane helix and in more N-terminally located helices can be detected already at the point when the C-terminal helix partitions from the translocon into the membrane. Our findings pinpoint the earliest steps of tertiary structure formation and open up possibilities to study the cotranslational folding of polytopic membrane proteins.protein folding | membrane insertion | helix-helix interactions

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“…Helix-bundle membrane proteins display a large number of inter-helical contacts that are often formed between polar or even charged residues (25)(26)(27)(28). We noticed that some TM segments of GPCRs that are under study in our lab do not display favorable energies for full membrane insertion (29,30), an observation not unlike that reported for individual TMs of bacteriorhodopsin (31).…”

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confidence: 55%

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates

Poms

Ansorge

Martínez-Gil

et al. 2016

Journal of Biological Chemistry

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Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot et al., Biochemistry 29(1990), 4031) is postulated to proceed in 2 steps: Partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin et al., Biophys. J. 96(2009), 3187), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123 and TM127 adopt predominantly nativelike topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior. (7), and the structure of a GPCRarrestin complex was solved (8).While our knowledge of the structure of GPCRs in various states and their mode of activation and desensitization is rapidly increasing, detailed information on their folding pathways is still lacking. The popular refined two-stage model from Popot and Engelman postulates that secondary structure forms when the peptide chain partitions into the membranewater interface (9-11). However, proteins destined for membrane insertion are generally subjected to the concerted action of translating ribosomes in the cytoplasm and translocon complexes located in the endoplasmic reticulum (ER) of eukaryotes or in the plasma membrane of bacteria (12). In order to traffic proteins to membranes in most cells the signal-recognition particle targets the nascent chains emerging from the ribosome tunnel to the translocon complex (13 (12,(18)(19)(20). Folding of polytopic membrane proteins is the result of a series of events that include helixinsertion into the hydrophobic core and sequestering of loop-sequences into cytosolic or extracellular space (11,21). The timing of the chain insertion and the localization of TM helices would be expected to be a consequence of the amino acid sequence and the interaction of the growing polypeptide chain with the membrane. Recently, however, the first evidence was obtained that helices might change their locat...

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Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

Cited by 32 publications

References 50 publications

The Tobacco mosaic virus Movement Protein Associates with but Does Not Integrate into Biological Membranes

The Tobacco mosaic virus Movement Protein Associates with but Does Not Integrate into Biological Membranes

Cotranslational folding of membrane proteins probed by arrest-peptide–mediated force measurements

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates

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