The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution

Carlson, Michael Luke; Young, John William; Zhao, Zhiyu; Fabre, Lucien; Jun, Daniel; Li, Jianing; Li, Jun; Dhupar, Harveer Singh; Wason, Irvinder Singh; Mills, Allan; Beatty, J. Thomas; Klassen, John S.; Rouiller, Isabelle; Duong, Franck

doi:10.7554/elife.34085

Cited by 137 publications

(184 citation statements)

References 47 publications

Supporting

Mentioning

167

Contrasting

Order By: Relevance

“…We then reconstituted the complex into peptidisc (Supplementary Fig. 1), a recently developed short peptide that can stabilize the transmembrane region of membrane proteins in the absence of detergent 17 . The sample reconstituted in the peptidisc behaved much better on EM grids and led to a 3D reconstruction of the PlexinC1/A39R dimeric complex with the C2 symmetry to an overall 3.1 Å resolution (Table 1 and Supplementary Figs.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Cryo-EM structure of the PlexinC1/A39R complex reveals inter-domain interactions critical for ligand-induced activation

et al. 2020

View full text Add to dashboard Cite

Plexins are receptors for semaphorins that transduce signals for regulating neuronal development and other processes. Plexins are single-pass transmembrane proteins with multiple domains in both the extracellular and intracellular regions. Semaphorin activates plexin by binding to its extracellular N-terminal Sema domain, inducing the active dimer of the plexin intracellular region. The mechanism underlying this activation process of plexin is incompletely understood. We present cryo-electron microscopic structure of full-length human PlexinC1 in complex with the viral semaphorin mimic A39R. The structure shows that A39R induces a specific dimer of PlexinC1 where the membrane-proximal domains from the two PlexinC1 protomers are placed close to each other, poised to promote the active dimer of the intracellular region. This configuration is imposed by a distinct conformation of the PlexinC1 extracellular region, stabilized by inter-domain interactions among the Sema and membraneproximal domains. Our mutational analyses support the critical role of this conformation in PlexinC1 activation.

show abstract

Section: Resultsmentioning

confidence: 99%

“…The peptidisc peptide was purchased from PEPTIDISC LAB (https://peptidisc.com). The sequence of peptidisc peptide is FAEKFKEAVKDYFAKFWDPAAEKLKEAVKDYFAKLWD (Nterminus to C-terminus, single-letter amino acid code) 17 . The peptidisc peptide powder was dissolved at 2 mg/ml using 20 mM HEPES pH 7.5 and 150 mM NaCl.…”

Section: Articlementioning

confidence: 99%

Cryo-EM structure of the PlexinC1/A39R complex reveals inter-domain interactions critical for ligand-induced activation

et al. 2020

View full text Add to dashboard Cite

show abstract

“…In membrane-embedded formulations, this immunodominant neoepitope would be protected by the bilayer. Additional prevention of off-target immune responses against the scaffold protein can also be minimized with less immunoreactive peptide scaffolds, such as A22 (Kuai et al, 2017;Kuai et al, 2018) and peptidisc (Carlson et al, 2018). The third important factor in Env-nanodisc immunization would be the capacity to present the full set of quaternary epitopes and which more closely resemble the corresponding epitopes presented on native virus particles.…”

Section: Discussionmentioning

confidence: 99%

“…In nanodiscs, a complete detergent removal is done over ~48 hours in presence of MSP1D1 scaffold protein, leading to a stable lipid bilayer encircled by the scaffold. The peptidisc approach follows same principles but scaffold is now a short bi-helical, engineered peptide (Carlson et al, 2018). In the lipid bicelle approach, similar detergent removal leads to heterogeneously sized bicelles capped by lipid molecules and/or scaffold.…”

Section: Env Incorporation Into Detergentlipid Micelles Bicelles Andmentioning

confidence: 99%

HIV-1 Envelope and MPER antibody structures in lipid assemblies

Rantalainen

Berndsen

Antanasijevic

et al. 2019

Preprint

View full text Add to dashboard Cite

SummaryStructural and functional studies of HIV Env as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Thus, most structural studies have utilized soluble forms where the regions C-terminal to the ectodomain are deleted. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstituted a full-length Env clone into a nanodisc with MSP1D1 scaffold, complexed it with an MPER targeting antibody 10E8, and structurally defined the full quaternary epitope of 10E8 consisting of lipid, MPER and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapted the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with peptidisc scaffold.

show abstract

“…Expression and purification of SecYEG were performed from the plasmid pBad22 his-EYG (pEYG) as described previously (23,(27)(28)(29). Inner membrane vesicles (IMVs) containing overexpressed SecYEG were prepared from the E. coli strain KM9 as described previously (23,30).…”

Section: Protein Production and Purificationmentioning

confidence: 99%

Investigating the stability of the SecA–SecYEG complex during protein translocation across the bacterial membrane

Young

Duong

2019

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Edited by Chris WhitfieldDuring posttranslational translocation in Escherichia coli, polypeptide substrates are driven across the membrane through the SecYEG protein-conducting channel using the ATPase SecA, which binds to SecYEG and couples nucleotide hydrolysis to polypeptide movement. Recent studies suggest that SecA is a highly dynamic enzyme, able to repeatedly bind and dissociate from SecYEG during substrate translocation, but other studies indicate that these dynamics, here referred to as "SecA processivity," are not a requirement for transport. We employ a SecA mutant (PrlD23) that associates more tightly to membranes than WT SecA, in addition to a SecA-SecYEG crosslinked complex, to demonstrate that SecA-SecYEG binding and dissociation events are important for efficient transport of the periplasmic protein proPhoA. Strikingly however, we find that transport of the precursor of the outer membrane protein proOmpA does not depend on SecA processivity. By exchanging signal sequence and protein domains of similar size between PhoA and OmpA, we find that SecA processivity is not influenced by the sequence of the protein substrate. In contrast, using an extended proOmpA variant and a truncated derivative of proPhoA, we show that SecA processivity is affected by substrate length. These findings underscore the importance of the dynamic nature of SecA-SecYEG interactions as a function of the preprotein substrate, features that have not yet been reported using other biophysical or in vivo methods. Figure 5. Effect of the substrate leader peptide on SecA processivity. A, cartoon representations of proPhoA and the pOA ss -PhoA fusion protein. The leader peptide of proOmpA (red) was fused to the mature sequence of PhoA (blue). The molecular masses of each protein are indicated on the right. B, pOA ss-PhoA was fluorescently labeled and employed in translocation assays with SecYEG IMVs as described in Fig. 1C. FL, full-length. C, the amounts of fully translocated products and translocation intermediates were quantified as described for Fig. 1.

show abstract

The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution

Cited by 137 publications

References 47 publications

Cryo-EM structure of the PlexinC1/A39R complex reveals inter-domain interactions critical for ligand-induced activation

Cryo-EM structure of the PlexinC1/A39R complex reveals inter-domain interactions critical for ligand-induced activation

HIV-1 Envelope and MPER antibody structures in lipid assemblies

Investigating the stability of the SecA–SecYEG complex during protein translocation across the bacterial membrane

Contact Info

Product

Resources

About