New method allows analysis of proteins during dynamic processes on biological membranes

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Much of the biologically relevant process takes place at the membrane level. Studying the dynamics of these processes in real time and without disturbing the biological system remains a major methodological challenge. A team led by Petra Schwille, director of the Max Planck Institute of Biochemistry, and Nikolas Hundt of the Ludwig-Maximilians-Universität München has now developed a new method for this purpose: Mass-Sensitive Particle Tracking (MSPT). Using MSPT, the movements and reactions of individual unlabeled proteins on biological membranes can be determined solely by their mass.

Cellular processes on membranes are often rapid and short-lived. The molecules assemble briefly, separate again, interact with different partners, and move along or across the membrane. It is therefore important not only to study static snapshots of these processes, but also to understand their dynamics. But how do you get there methodically? Petra Schwille from the Max Planck Institute of Biochemistry and Nikolas Hundt from the Ludwig Maximilians University have developed with their team the Mass-Sensitive Particle Tracking – MSPT method, which makes it possible to analyze proteins during dynamic processes on membranes.

The starting point for biophysicists was recent advances in mass photometry, which could already be used to determine the molecular mass of unlabeled molecules in solution. The novelty of MSPT is that the dynamics of membrane-associated proteins can now be followed in their biologically plausible environment. In this process, individual proteins are identified by their molecular mass without the need for labeling.

Frederik Steiert, one of the first authors of the publication, says: “We can now follow directly on biological membranes the mass of individual proteins, how they move and how they interact. This allows us to study the dynamics of biological systems in more detail. . “The analysis of dynamic processes is particularly important in biology because many membrane-level processes are transient.

Determination of mass by light scattering

What principles is the new method based on? When light hits a particle, the light is scattered. The intensity of the scattered light depends on the mass of the particle. Videos in which individual proteins on membranes are made directly visible are recorded under a microscope. Using analysis software, these proteins can be tracked and their diffusion signal, and therefore their mass, can be determined. This is currently possible for proteins with a molecular weight of at least 50 kDa, that is to say for a large part of all known proteins. Another advantage of the new MSPT method is that proteins do not need to be labeled. Labeling can be accomplished, for example, by attaching fluorescent labels to molecules. However, the labeling presents the risk that the proteins may be altered in their function or that the fluorescent markers may turn white during the experiment. By using MSPT, on the other hand, methodological problems that may arise from labeling are avoided.

MinDE protein system

To demonstrate the method’s potential for biological questions, the biophysicists used an established system from Schwille’s laboratory: the bacteria’s MinDE protein system. Escherichia coli (E. coli). The MinD and MinE proteins are involved in E. coli cell division.

The method allows us to characterize properties of dynamical systems that were not previously measurable. This allowed us not only to verify the findings made on the Min system, but also to gain new knowledge. “

Tamara Heermann, first author

Using MSPT, the team was able to show that the MinD protein complexes are larger than initially thought. In addition, the experiments provide initial information that MinE can act as a connecting element for MinD proteins and thus can initiate membrane release of larger complexes.

As reported in the new article, MSPT provides valuable information to elucidate dynamic processes at the level of biological membranes. However, researchers are continuously working on improving the method even further. In the future, the method should also be applicable to integral membrane proteins and should allow the detection of even smaller proteins.

Source:

Journal reference:

Heermann, T., et al. (2021) Monitoring of mass-sensitive particles to elucidate the membrane-associated MinDE reaction cycle. Natural methods. doi.org/10.1038/s41592-021-01260-x.

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