Membrane proteins

Know your protein – inside out

Challenges in studying membrane proteins

Up to a third of all human genes encode membrane proteins — showing both their variety and significance. Mutations in membrane proteins cause many common human diseases like heart disease, neurological diseases, cancer, and cystic fibrosis, making them an extremely important target for drug development. In fact, over 50% of current small molecule drugs target membrane proteins.

Despite their prevalence and importance, less than 1% of high-resolution protein structures are membrane proteins. Expression, purification and stabilization of membrane proteins is time consuming and difficult, and even if achieved, the immobilization required for many interaction analysis technologies risks damaging the precious protein. This wide array of challenges therefore begs the question of whether it could be possible to study membrane protein structural stability, folding, oligomerization, binding and function all without purification?

How can we help?

We built our Fluidity One-M instrument with your challenges in mind. Analysis in-solution on our single-use microfluidic channels that are optimized for running crude membrane preparations eliminates artefacts.

The smart assistant – Fluidity Insight – with advanced machine learning experiment guidance, will ensure you’re always on the right path.

True size

Work directly with full-length protein. See how ligand binding affects conformation by observing size changes.

True environment

Measure protein in its membrane environment and skip the hassle of optimizing expression, purification and detergent screening.

True insights

Quantify expression level with certainty and understand binding affinity in your target’s native context.

Application Note

Purification-free affinity and concentration measurement of membrane-protein targets

Here, we introduce an integrated preparative and analytical method that enables researchers to simultaneously measure both the concentration of an endogenous membrane-protein target and its binding affinity to a specific ligand in a native lipid-bilayer environment directly extracted from crude cellular membranes.

True size

Glyco-DIBMA solubilizes lipid vesicles to yield small and stable nanodiscs that preserve the bilayer architecture and are compatible with chemical protein-conjugation techniques. In this study, Glyco-DIBMA was used to solubilize the E. coli membrane expressing his-tagged voltage-gated K+ channel protein KvAP. The nanodiscs were then then labelled with ATTO-488 and the sizes of free dye and nanodisc-embedded ATTO-488-KvAP were measured using Microfluidic Diffusional Sizing.

Read more from the original article.

 

True environment

One of the most challenging factors when working with membrane proteins is obtaining soluble and properly folded protein. Working directly with full-length protein and eliminating the need for detergents greatly simplifies the membrane protein workflow while simultaneously precluding the introduction of artefacts. In our application note, we produce crude membrane protein libraries that can be directly interrogated in binding studies by solubilizing complete cell membranes into nanodiscs. Using MDS, we can measure the binding of a labeled antibody probe to membrane proteins embedded in nanodiscs.

True insights

In this example, the copy number of the membrane protein HER2 present in the starting cell membrane preparation was determined by titrating the sample against different concentrations of labelled target. This result greatly improves upon the current stratification of HER2 levels on cancer cells into broad categories of negative (0, 1+), positive (3+), or unresolved (2+).

Learn how your peers studied membrane proteins with MDS

Determine sizes of mixed lipid protein particles

Membrane proteins are highly sensitive to their environment, and so exchanging their native lipid for synthetic detergents can negatively impact their structure and function. Danielczak et al. established a new polymer for generation of lipid nanodiscs that is highly biologically compatible. The nanodiscs formed using the new polymer show consistent size with a range of synthetic lipids and embedded proteins can be fluorescently tagged in situ highlighting the consistency and robustness of the nanodisc polymer.

Danielczak, Bartholomäus, et al. “A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs” Nanoscale 14 (2022): 1855-1867

Fluidic Sciences Ltd