Introduction to Membrane Proteins

Chiu, Mark L.

doi:10.1002/0471140864.ps2901s67

Cited by 6 publications

(4 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

Section: Introductionmentioning

confidence: 99%

“…Membrane proteins are important actors in cross‐membrane cell physiology and fulfill a variety of functions ranging from cellular signaling to energy generation and transport 1,2 . They cover about 20–50% of prokaryotic and eukaryotic genomes, 2 and constitute more than 50% of all approved drug targets 3 . Many membrane proteins are involved in the pathogenesis of several severe diseases (e.g., cystic fibrosis) due to their misfolding from sequence mutations 4 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Heat treatment of thioredoxin fusions increases the purity of α‐helical transmembrane protein constructs

et al. 2021

View full text Add to dashboard Cite

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α‐helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute‐timescale heat treatment of Trx‐tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single‐step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Heat treatment of thioredoxin fusions increases the purity of α‐helical transmembrane protein constructs

et al. 2021

View full text Add to dashboard Cite

show abstract

“…In addition to the requirement for relatively large amounts of protein, there is also a need to keep the protein folded and functional in native membrane lipid mimics (detergents) over a period of days. A tremendous amount of work has gone into obtaining large amounts of proteins mainly by optimizing heterologous expression in insect, yeast, mammalian, and bacterial systems, and in parallel, developing and using detergents that are mild and at the same time efficient in solubilizing membranes [6,7]. Until recently, the major goals of these efforts have been focused on structure determination, and indeed, a recent flurry of papers on structures of various GPCRs bodes well that protein expression and obtaining sufficient amounts of proteins will not be limiting [8-10], and will become less cumbersome, setting the stage for future ITC studies of this highly challenging but important class of proteins.…”

Section: Introductionmentioning

confidence: 99%

Isothermal titration calorimetry of membrane proteins — Progress and challenges

Rajarathnam

Rösgen

2014

Biochimica et Biophysica Acta (BBA) - Biomembranes

View full text Add to dashboard Cite

Summary Integral membrane proteins, including G protein-coupled receptors (GPCR) and ion channels, mediate diverse biological functions that are crucial to all aspects of life. The knowledge of the molecular mechanisms, and in particular, the thermodynamic basis of the binding interactions of the extracellular ligands and intracellular effector proteins is essential to understand the workings of these remarkable nanomachines. In this review, we describe how isothermal titration calorimetry (ITC) can be effectively used to gain valuable insights into the thermodynamic signatures (enthalpy, entropy, affinity, and stoichiometry), which would be most useful for drug discovery studies, considering that more than 30% of the current drugs target membrane proteins.

show abstract

“…Membrane protein genes represent approximately a third of all the genes identified so far in humans. , Membrane proteins (MPs) have a variety of functions ranging from membrane transport to cell signal transduction, and many of them are molecular targets for drugs and low molecular ligands . Therefore, new analytical methods are under development to enable better characterization of MPs in terms of their structure and function.…”

mentioning

confidence: 99%

Electrochemical Platform for the Detection of Transmembrane Proteins Reconstituted into Liposomes

et al. 2016

View full text Add to dashboard Cite

The development of new methods and strategies for the investigation of membrane proteins is limited by poor solubility of these proteins in an aqueous environment and hindered by a number of other problems linked to the instability of the proteins outside lipid bilayers. Therefore, current research focuses on an analysis of membrane proteins incorporated into model lipid membrane, most frequently liposomes. In this work, we introduce a new electrochemical methodology for the analysis of transmembrane proteins reconstituted into a liposomal system. The proposed analytical approach is based on proteoliposomal sample adsorption on the surface of working electrodes followed by analysis of the anodic and cathodic signals of the reconstituted proteins. It works based on the fact that proteins are electroactive species, in contrast to the lipid components of the membranes under the given experimental conditions. Electroanalytical experiments were performed with two transmembrane proteins; the Na(+)/K(+)ATPase that contains transmembrane as well as large extramembraneous segments and the mitochondrial uncoupling protein 1, which is a transmembrane protein essentially lacking extramembraneous segments. Electrochemical analyses of proteoliposomes were compared with analyses of both proteins solubilized with detergents (C12E8 and octyl-PoE) and supported by the following complementary methods: microscopy techniques, protein activity testing, molecular model visualizations, and immunochemical identification of both proteins. The label-free electrochemical platform presented here enables studies of reconstituted transmembrane proteins at the nanomolar level. Our results may contribute to the development of new electrochemical sensors and microarray systems applicable within the field of poorly water-soluble proteins.

show abstract

Introduction to Membrane Proteins

Cited by 6 publications

References 63 publications

Heat treatment of thioredoxin fusions increases the purity of α‐helical transmembrane protein constructs

Heat treatment of thioredoxin fusions increases the purity of α‐helical transmembrane protein constructs

Isothermal titration calorimetry of membrane proteins — Progress and challenges

Electrochemical Platform for the Detection of Transmembrane Proteins Reconstituted into Liposomes

Contact Info

Product

Resources

About