How to utilize Grating Coupled Interferometry (GCI) for complex membrane proteins
Anesthesia may have revolutionized the world of medicine, but recovering from being ‘put under’ is never an enjoyable experience. After all, the medication is powerful enough to stop nerves sending pain signals by targeting membrane proteins in the brain – so it’s no surprise if it takes time before we feel like ourselves again!
Why research membrane proteins?
Anesthesia isn’t the only medication with potential for improvement. Across several treatments, the search is on for more effective, patient-friendly options. But in many cases, the drug interactions involved aren’t fully understood.
A large proportion of drugs target membrane proteins: proteins found within cell membranes, that are often involved in signaling, transport, or cellular communication. There are several types of membrane proteins: transporters, ion channels, and in particular, G-protein-coupled receptors (GPCRs). Around 30% of all prescription pharmaceuticals on the market target GPCRs.
To support drug development, researchers are working hard to build a clearer picture of the interactions between drugs and the human body. And that’s where accurate and reliable biophysical characterization comes in. With GCI technology in particular shining light on targeting these complex membrane proteins.
From binding affinity to kinetic rate constants
When drugs interact with membrane proteins, they affect cell signaling cascades to elicit biochemical changes. These biochemical changes can impact disease, or in the case of anesthetics, block signals to the nervous system. We can tweak, or ‘inhibit’ these biochemical changes by targeting membrane proteins with novel compounds to achieve a more desirable outcome.
Understanding biomolecular interaction analysis
- Binding affinity (KD) – how tightly the compound interacts with the target.
Traditionally, binding affinity has been a key indicator of a drug’s effectiveness. Now though, scientists can also garner even greater insight through kinetic rate constants.
- Kinetic rate constants (ka, kd) – how quickly a compound associates with it’s targets and how quickly the it’s dissociates.
Understanding the kinetic rate constants enables a more comprehensive overview of the compounds effects than binding affinity alone.
The challenges of complex membrane protein analysis
The bad news? The binding affinity and interaction kinetics of membrane proteins – particularly GPCRs are notoriously difficult to study. When GPCRs are extracted from a cell membrane, their high hydrophobicity leads to rapid disassembly, rendering the sample useless for analysis.
Isotopic labelling vs biosensor based methods
Isotopic labelling: To prevent sample loss, stable isotopes can be used to ‘label’ GPCRs, but since these isotopes are very expensive, ‘label-free’ biosensor-based methods like surface plasmon resonance (SPR) are often preferred.
However, developing robust assays with required sensitivity using traditional biosensor-based methods comes with it’s challenges. Membrane protein samples require a particularly high level of precision and instrument sensitivity. Furthermore, complex, heterogeneous samples containing a variety of substances can often clog the instrument’s microfluidics and block critical observations. Time for Malvern Panalytical’s WAVEsystem, a Creoptix® GCI technology to step up!
How GCI outperforms other interaction analysis
The WAVEsystem uses grating-coupled interferometry (GCI), a highly sensitive, label-free, biosensor-based method. In GCI (as with other biosensor-based techniques) the GPCRs can be kept in a lipid environment resembling the cell during the target capture onto the sensor surface. However, GCI’s detection principle is different: it measures changes in interference patterns caused by molecular binding events on a nanostructured grating surface.
This detection principle makes GCI inherently sensitive. In combination with no-clog microfluidics, this sensitivity makes it possible to analyze complex targets like GPCRs. GCI’s sensitivity also means that even weak interactions – such as those of protein fragments – can be analyzed, and ensures researchers can trust their results even in suboptimal conditions.
Figure 1: Results from analysis of GPCRs from crude membrane extracts using GCI. (A) The Nterminal-AVI target GPCR (red), the Cterminal-AVI target GPCR (green), and the negative control Nterminal-AVI GPCR (blue) were injected onto a 4PCP-STA WAVEchip® at 10 µl per minute.(B) Five concentrations (0.96 nM – 3 µM) of the anti-target-GPCR nanobody were then injected in duplicate for 180 seconds at 45 µl per minute.
Read more about this crude membrane extract example in our whitepaper here.
Kinetics in hours, not days
As well as outperforming conventional biosensor-based methods for kinetic interaction analysis, WAVEsystem brings all kinds of other advantages.
Take the WAVEchip® technology, which combines the microfluidics and biosensor in a single disposable sensor chip, compatible with crude samples like serum, plasma, or membrane extracts. By placing valves downstream in the instrument instead of having microvalves near the chip, the sensor chip design enables ultra-fast transition times and reliable determination of off-rates, enabling the kinetic study of weakly binding fragments.
But that’s not all. The platform’s intuitive software and ability to handle heterogeneous samples means there’s no need for a lengthy sample-purification process – turning a two-day task into an hour’s work. In turn, WAVEsystem enables high throughput interactions analysis necessary to scale up biosensor drug discovery.
Makin’ WAVEs
Bringing together the most accurate, efficient, and sensitive membrane protein analysis methods with a variety of user-friendly functionalities, WAVEsystem is certainly making waves in the world of drug discovery. With powerful analytical platforms like this, maybe a fatigue-free anesthetic is just around the corner…
Want to learn more? Sign up for our upcoming webinar:
‘Fragment screening of G-protein-coupled receptors using GCI technology’
Click Here to Register
See you on April 9, 2024, at 10:00 EST/15:00 CET!