Subnanometer-Precision Measurements of Transmembrane Motions of Biomolecules in Plasma Membranes Using Quenchers in Extracellular Environment

Abstract

It is a big challenge to measure position changes of biomolecules in the direction normal to the plasma membranes of living cells. We developed a one donor-multiple quenchers Fӧrster resonance energy transfer method by using non-fluorescent quenchers in the extracellular environment. It senses subnanometer position changes of a fluorophore-labeled biomolecule in the plasma membrane. The method was validated by monitoring flip-flops of individual lipid molecules incorporated in plasma membranes. We studies membrane perforation by a host defense peptide from the extracellular side and found that the pore-forming peptide is dynamic, switching among different insertion depths. The method is especially useful in studying interactions of membrane proteins with the inner surfaces of plasma membranes. Our method will find wide applications in systematic analysis of fundamental cellular processes at plasma membranes.

Understanding Förster Resonance Energy Transfer in the Sheet Regime with DNA Brick-Based Dye Networks

“…An understanding of FRET in the sheet regime and how it is affected by various nonidealities is not just an academic exercise, but can inform real-world applications. Sheet regime effects become critical for quantitatively interpreting FRET microscopy of cellular membranes where one typically monitors dye-labeled D moieties interacting with membranes labeled with numerous lipophilic A dyes (or displaying some endogenous receptor/protein that can similarly act as the A sheet) in order to gather information about ligand–receptor interaction dynamics, vesicle formation, membrane biomolecular sensing events, etc . ,− The density of fluorophores in such confines can make interpretation of ET and subsequent distance estimates complex. Moreover, it is in just such situations where changing the 1/ r α exponent value from 6 to 4 can significantly alter estimated separation distances and assumptions of viable FRET sensing ranges.…”

“…Our goal in this paper is to better understand some of the key variables that enable Förster transfer in the sheet regime in real systems. Our focus is on systems of discrete donors and acceptors and not on physically continuous systems such as graphene, metallic absorbers, or surfaces, other 2D materials, or larger (metallic) plasmonic nanoparticle surfaces whose optoelectronic properties are still not fully characterized and where more complex processes may potentially be involved. − Beyond designing improved FRET networks, this work is also relevant to FRET-based applications such as fluorescent imaging and characterization of cellular membranes and probing of ligand–receptor interactions. ,− …”

Cell membrane-coated nanoparticles: a novel multifunctional biomimetic drug delivery system

“…The uniformity of encapsulation is a significant factor when evaluating a hybrid cell membrane. Förster resonance energy transfer (FRET) can visualize the fusion process and uniformity [ 27 , 92 , 93 ]. For instance, during the preparation of RBC–platelet hybrid membranes [ 52 , 91 ], the intensity of dye on platelets interacting with RBCs would increase on increasing the RBC input, whereas the intensity of dye interacting with platelets would decrease, indicating the dispersion and fusion of the two membrane materials.…”

Single-molecular methodologies for the physical biology of protein machines

“…Finally, if necessary, the observations can be confirmed on real cells to see if the dynamics and functions of the proteins of interest would be modified by other proteins, by which corporation of proteins could be unveiled. [69] Force Nucleus Cytoplasm…”