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Related Concept Videos

Introduction to Membrane Proteins01:16

Introduction to Membrane Proteins

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The cell membrane, or plasma membrane, is an ever-changing landscape. It is described as a fluid mosaic where various macromolecules are embedded in the phospholipid bilayer. Among the macromolecules are proteins. The protein content varies across cell types. For example, mitochondrial inner membranes contain ~76% protein content, while myelin contains ~18% protein content. Individual cells contain many types of membrane proteins—red blood cells contain over 50—and different cell...
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Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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Insertion of Single-pass Transmembrane Proteins in the RER01:26

Insertion of Single-pass Transmembrane Proteins in the RER

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Integral membrane proteins are proteins adhered to the lipid bilayer of a cell organelle or membrane. They can be of two types: transmembrane integral proteins that span the lipid bilayer and monotopic proteins that are attached to either side of the membrane but do not pass through it.
Integral transmembrane proteins possess transmembrane and extra membrane domains. The transmembrane domains are primarily made of 20-25 hydrophobic amino acids arranged in a helical secondary confirmation. These...
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SNAREs and Membrane Fusion01:43

SNAREs and Membrane Fusion

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Once a transport vesicle has recognized its target organelle, the vesicular membrane needs to fuse with the target membrane to unload the cargo. Transmembrane proteins called SNAREs present on organelle membranes and their vesicles, mediate vesicle fusion.
SNAREs exist in pairs that symmetrically interact and catalyze the fusion of the lipid bilayers in vesicle and target organelle. v-SNARE in the vesicle membrane are single polypeptide chains that bind to a complementary t-SNARE, composed of 2...
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Tail-anchoring of Proteins in the ER Membrane01:45

Tail-anchoring of Proteins in the ER Membrane

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Tail-anchored, or TA, proteins are estimated to make up to 3-5% of membrane proteins found in the eukaryotic cell. Such proteins have a single transmembrane domain located approximately 30 amino acid residues upstream from the C-terminal end. As a result, the signal recognition particle (SRP) cannot guide a TA protein to the ER membrane for cotranslational insertion. Hence, they are integrated into the ER membrane post-translationally using their C-terminal end as the anchor. TA proteins...
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Protein Transport into the Inner Mitochondrial Membrane01:34

Protein Transport into the Inner Mitochondrial Membrane

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Nuclear encoded mitochondrial precursors are imported to the inner membrane in a multistep process involving two separate translocons, TIM22 and TIM23. TIM23 is a cation-selective pore that remains closed by the N terminal segment of the protein. Negative charges on the TIM23 act as a receptor for the incoming precursor, pulling the positively charged matrix-targeting sequence for peptide insertion and translocation.
Transport of mitochondrial precursors across the TIM23 channel is driven by...
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Synthesis of an Intein-mediated Artificial Protein Hydrogel
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Semisynthesis of Membrane-Attached Proteins Using Split Inteins.

Stefanie Hackl1, Alanca Schmid1, Christian F W Becker2

  • 1Department of Chemistry, Institute of Biological Chemistry, University of Vienna, Waehringer Str. 38, 1090, Vienna, Austria.

Methods in Molecular Biology (Clifton, N.J.)
|October 8, 2016
PubMed
Summary

Researchers developed a method using split inteins for site-selective protein lipidation. This technique attaches lipid anchors to proteins of interest (POIs) via protein trans-splicing (PTS) for studying membrane association.

Keywords:
Green fluorescent proteinLipid-coated particlesLiposomesMembrane-associated proteinsPrion proteinProtein semisynthesisProtein trans-splicingSplit inteinsSynthetic membrane anchor

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Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo
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Area of Science:

  • Biochemistry and Molecular Biology
  • Protein Engineering
  • Cell Biology

Background:

  • Site-selective protein lipidation is crucial for understanding protein biophysics and native function.
  • Membrane association significantly impacts protein properties and cellular roles.
  • Current methods for protein modification can be challenging and lack specificity.

Purpose of the Study:

  • To develop a novel method for site-selective C-terminal attachment of lipid-modified peptides to proteins of interest (POIs).
  • To enable the study of protein-membrane interactions using protein trans-splicing (PTS) and split inteins.
  • To provide a versatile platform for lipidating various POIs, including challenging targets like the prion protein.

Main Methods:

  • Utilized split inteins for protein trans-splicing (PTS) to ligate protein of interest (POI) with a modified peptide.
  • Expressed POI fused to an N-terminal split intein segment.
  • Synthesized a C-terminal split intein segment, chemically ligated to a dual-lipidated membrane anchor peptide.

Main Results:

  • Successfully demonstrated the C-terminal attachment of a lipidated membrane anchor (MA) peptide to target proteins.
  • Validated the method using the prion protein (PrP) and enhanced green-fluorescent protein (eGFP) as model systems.
  • Showcased the applicability of split intein systems from Synechocystis spp. and Nostoc punctiforme.

Conclusions:

  • Split intein-mediated protein trans-splicing offers a robust and site-selective approach for protein lipidation.
  • This method facilitates the investigation of protein-membrane interactions and the biophysical properties of lipidated proteins.
  • The technique is adaptable for various proteins of interest, expanding possibilities in biochemical and cell biology research.