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Related Concept Videos

Single-pass Transmembrane Proteins01:25

Single-pass Transmembrane Proteins

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Integral membrane proteins are tightly associated with the cell membrane and play a crucial role in cell communication, signaling, adhesion, and transport of the molecules. Some integral membrane proteins are present only in the membrane monolayer. For example, the enzyme fatty acid amide hydrolase is present in the cytoplasmic side of the membrane monolayer. In contrast, another type of integral membrane protein, also known as a transmembrane protein, spans across the membrane. Transmembrane...
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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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Insertion of Multi-pass Transmembrane Proteins in the RER01:29

Insertion of Multi-pass Transmembrane Proteins in the RER

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The rough ER membrane synthesizes, assembles, and embeds transmembrane proteins in diverse topologies. These proteins function as transporters or channels and can remain in the ER membrane or are sent to the Golgi complex, lysosome, and cell membrane.
The multipass transmembrane proteins are the type IV integral membrane proteins with multiple topogenic sequences determining their spatial arrangement in the ER membrane. Nearly all multipass proteins lack a cleavable signal sequence and use...
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Factors Affecting Solubility04:01

Factors Affecting Solubility

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Compared with pure water, the solubility of an ionic compound is less in aqueous solutions containing a common ion (one also produced by dissolution of the ionic compound). This is an example of a phenomenon known as the common ion effect, which is a consequence of the law of mass action that may be explained using Le Chȃtelier’s principle. Consider the dissolution of silver iodide:
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Solubility Equilibria03:07

Solubility Equilibria

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Solubility equilibria are established when the dissolution and precipitation of a solute species occur at equal rates. These equilibria underlie many natural and technological processes, ranging from tooth decay to water purification. An understanding of the factors affecting compound solubility is, therefore, essential to the effective management of these processes. This section applies previously introduced equilibrium concepts and tools to systems involving dissolution and precipitation.
The...
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Multi-pass Transmembrane Proteins and β-barrels01:09

Multi-pass Transmembrane Proteins and β-barrels

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In multi-pass transmembrane proteins, the polypeptide chain crosses the membrane more than once. The transmembrane polypeptide chain either forms an α-helix or β-strand structure. α-Helix containing multi-pass transmembrane proteins are ubiquitous, whereas β-strand containing ones are mainly found in gram-negative bacteria, mitochondria, and chloroplasts.
α-Helix containing multi-pass transmembrane proteins
Multi-pass transmembrane proteins such as...
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Related Experiment Video

Updated: Jan 21, 2026

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
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CETSA beyond Soluble Targets: a Broad Application to Multipass Transmembrane Proteins.

Aarti Kawatkar1, Michelle Schefter1, Nils-Olov Hermansson2

  • 1Discovery Sciences, BioPharmaceutical R&D , AstraZeneca , Boston , United States.

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Live-cell cellular thermal shift assay (CETSA) successfully assesses target engagement for challenging membrane proteins. Modified protocols enable reliable analysis of diverse transmembrane proteins, offering insights into therapeutic action.

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Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
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Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
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Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
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Area of Science:

  • Biochemistry
  • Pharmacology
  • Cell Biology

Background:

  • Target engagement is crucial for understanding therapeutic mechanisms.
  • The cellular thermal shift assay (CETSA) is a label-free method for assessing target engagement in cells.
  • Integral multipass transmembrane proteins are important drug targets but challenging to study.

Purpose of the Study:

  • To demonstrate the application of live-cell CETSA to integral multipass transmembrane proteins.
  • To showcase the utility of CETSA for diverse membrane protein targets with varying stabilization behaviors.
  • To provide guidance for researchers conducting CETSA experiments on membrane proteins.

Main Methods:

  • Live-cell CETSA was applied to three distinct membrane proteins: TSPO, SERCA2, and PAR2.
  • Modified protocols involving detergent extraction post-heating were utilized.
  • Protein stabilization was assessed in response to compound treatment.

Main Results:

  • Robust stabilization of the outer mitochondrial protein TSPO was observed.
  • Modest stabilization was detected for SERCA2.
  • Atypical compound-driven stabilization was shown for the GPCR PAR2.
  • The modified CETSA protocol proved reliable for various membrane protein complexities.

Conclusions:

  • Live-cell CETSA is a versatile method applicable to a range of integral multipass transmembrane proteins.
  • Modified protocols enhance the reliability of CETSA for challenging membrane-bound targets.
  • Understanding distinct CETSA behaviors is essential for interpreting results with membrane proteins.