Webinar Monitoring of SMALP-nanodisc formation by MDS Published on December 14th, 2020 Styrene–maleic acid (SMA) copolymers have become popular tools in membrane-protein research as they extract both lipids and membrane proteins from cellular membranes while preserving the bilayer structure of the membrane in so-called nanodiscs. Microfluidic diffusional sizing (MDS) demonstrates how the SMA-to-lipid ratio controls styrene–maleic acid lipid particle (SMALP) size, which can be a crucial parameter in the characterization of embedded membrane proteins. Moreover, MDS offers the opportunity to measure nanodisc size as well as protein complex formation using the same sample. Nanodiscs are disc-shaped patches of lipid bilayers that are widely used to stabilize membrane proteins for functional and structural studies. A common method to prepare nanodiscs is based on the addition of styrene–maleic acid copolymers (SMAs) to intact cell membranes which spontaneously generates nanodiscs known as SMA–lipid particles. Transcript: Monitoring of SMALP-nanodisc formation by microfluidic diffusional sizing “Hello and welcome to this seminar series where we will be exploring the applications that have been completed using microfluidic diffusional sizing on the Fluidity One-W. My name is Haris Choudhery, I am the content marketing associate for Fluidic Analytics, and today I will be taking you through an application where fluorescently labeled lipids were used to observe SMALP-nanodisc formation using the Fluidity One-W instrument. Overview First, we will briefly cover the function of styrene-maleic acid (SMA) copolymers and how they have changed the way researchers are now investigating membrane-protein interactions. We will then discuss how microfluidic diffusional sizing, or MDS, on the Fluidity One-W was used to observe the onset of SMALP formation at low SMA-to-lipid ratios. From the measured hydrodynamic radius of these SMALP nanodiscs, we will show how the radius of the nanodiscs was inferred and in turn the number of POPC molecules that made up the nanodisc radius was calculated. Isolating membrane proteins for research Proteins embedded within lipid membranes are crucial biological gatekeepers that mediate the interactions between cells and their environment and control the passage of most molecules into and out of cells. To illustrate their importance, one quarter of the human genome encodes for membrane proteins, and yet, they represent two thirds of known drug targets. However, studying membrane proteins presents several challenges: they are not abundant and can be difficult to purify and characterize due to the requirement for their membrane-embedded regions to be protected from the aqueous environment. As a result, our limited knowledge of the structures and functions of membrane proteins is reflected in our incomplete understanding of the fundamental biological processes they govern and impairs our ability to develop treatments for a variety of diseases. Introduction to nanodiscs A promising tool in membrane research is the detergent-free solubilization of membrane proteins by styrene-maleic acid copolymers (SMAs). These amphipathic molecules can solubilize lipid bilayers to create small regions of lipid membrane, or nanodiscs, that are bounded by the polymer. Once formed, these nanodiscs are stable and readily handled, and so can, for example, be subjected to purification procedures. Thus, SMA lipid particles, or SMALPs are an excellent tool for membrane protein research as membrane proteins can be directly extracted from cells in a water-soluble form while conserving a patch of native membrane around them. By conserving the native structure of the membrane protein, researchers can more accurately characterize the interactions between these proteins and the wider cellular environment as well as identify small molecule or drug targets. Microfluidic diffusional sizing on the Fluidity One-W was used to observe the onset of SMALP formation at low SMA-to-lipid ratios. As the SMA-to-lipid ratio was increased, vesicles started to disintegrate, coinciding with the presence of large and potentially heterogenous SMALPs before yielding stable and consistent SMALPs as the SMA-to-lipid ratio was increased further. Fluidity One-W Before delving deeper into these results, I want to quickly take you through how MDS works I just want to take you through a few key features of the instrument itself. The size range the Fluidity One-W is able to measure is from 0.5nm to 20nm. This range covers most small molecules and dipeptides all the way up to approximately 15 megadaltons, so almost all of the protein species that might be found in a cell. It is compatible with green fluorophores, including Fluidiphore rapid amine 503, FITC, GFP or Alexa488 equivalent dyes. High sensitivity means that KD values for binding interactions in the pico molar range can be measured. Importantly, these measurements are carried out, fully in solution with no surface fixing and using just 5 ul of sample per measurement. What is Microfluidic Diffusional Sizing The following video explains how microfluidic diffusional sizing works. Fluorescence traces for LUVs As the sample passes through the chip, fluorescence intensities of the undiffused material and the diffused material, F-undiffused and F-diffused respectively, are recorded over time, resulting in fluorescence traces as shown on screen. Difference in signal ratios With these traces we can examine the degree of diffusion by using the signal ratio which is the diffused signal intensity divided by the undiffused signal intensity. Large particles diffuse slowly and so have a low signal ratio whereas smaller particles that diffuse more quickly have higher signal ratios. Examining the traces shown here, we can see that the intact LUVs gave an SR value of 0.15, Indicating low levels of diffusion were taking place and thus that these are large particles. In contrast, fluorescence traces of identical lipid mixtures in the presence of SMA yielded an SR value of approximately 0.75. Indicating higher levels of diffusion caused by the conversion of the large LUVs into much smaller SMALP nanodiscs. Measuring the size of SMALP nanodiscs To analyze how the ratio of SMA to lipid affects SMALP nanodisc size, fluorescently labeled LUVs at three different concentrations of total lipid (25, 50 and 100 μM) were mixed with increasing amounts of SMA. These three different experiments fall on a single curve when SR is plotted as a function of the SMA-to-lipid ratio showing that SMALP formation is dependent solely on the SMA-to-lipid ratio and not on the absolute concentrations. An increase in SR indicates that SMA extracts lipids from LUVs leading to formation of smaller particles that coexist with intact LUVs. It is known from the literature that the point of complete LUV solubilization, when no LUVs remain in solution, comes at an SMA to lipid ratio of about 0.15. However, as shown by our Fluidity One-W experiments, at this point of complete LUV solubilization, the average hydrodynamic radius, Rh, of the SMALP nanodiscs is 20 nm, and this decreases as more SMA is added to a final, stable Rh of 4.5 nm from a SMA to lipid ratio of 1. Based on the measured Rh, the radius of the nanodiscs can be determined based on the existing knowledge that POPC bilayers are 4 nm thick. Thus, the hydrodynamic radius values of 20 nm and 4.5 nm result in a nanodisc radius of 26 nm and 5 nm, respectively. With a known nanodisc radius it is possible to calculate the number of SMALP-embedded lipids making up the nanodisc. Therefore, in a larger SMALP nanodisc with an Rh of 20 nm, there are approximately 60 POPC lipid molecules between the center and the edge of the nanodisc, whereas only 11 POPC molecules make up the radius of a nanodisc with an Rh of 4.5 nm. Notably, the presence of a membrane protein would further reduce the number of nanodisc-embedded lipids which should be kept in mind if the lipid environment is a relevant consideration for downstream analysis. Conclusion Microfluidic diffusional sizing is an ideal tool to monitor SMALP nanodisc formation using minute amounts of sample. According to our data, SMALP nanodiscs vary in their average hydrodynamic radii from 20 nm to 4.5 nm depending on the SMA-to-lipid ratio. This change in size will significantly alter the lipid environment around any embedded proteins and so should be considered by researchers working on such systems. In the future similar experiments could be performed using a fluorescently labeled protein as a probe. This approach would enable researchers to confirm nanodisc sizes as well as measure complex formation of the embedded membrane protein using the same nanodisc sample. About Us Before I leave you, I just want to give you a brief overview of Fluidic Analytics. We were founded in 2013 by Prof Tuomas Knowles and Andrew Lynn. Our headquarters are in Cambridge, but we have offices in Zurich and Boston. We are developing instruments for protein analysis because we believe proteins are the key players in the biological stage and that science will benefit from new and distinct protein analytical technologies. Our first product is the Fluidity One‑W described here today and is available for purchase or demo today. Thank you for listening, if you would like to discuss any possible applications or like to demo the Fluidity One-W in your own lab, feel free to contact one of our scientists at firstname.lastname@example.org.