Taking Care of Bystander FRET in a Crowded Cell Membrane Environment

Abstract

Oligomerization of membrane proteins is a key event in cell signaling, and yet it is challenging to explore experimentally due to the complexity associated with cell membranes (1Ganguly S. Clayton A.H.A. Chattopadhyay A. Organization of higher-order oligomers of the serotonin1A receptor explored utilizing homo-FRET in live cells.Biophys. J. 2011; 100: 361-368Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The article by King et al. (2King C. Sarabipour S. Hristova K. et al.The FRET signatures of non-interacting proteins in membranes: simulations and experiments.Biophys. J. 2014; 106: 1309-1317Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) in this issue of the Biophysical Journal describes a strategy for avoiding a major problem in exploring interaction between membrane proteins utilizing fluorescence resonance energy transfer (FRET). The problem has to do with FRET arising from membrane proteins that do not interact, but still give rise to FRET because they happen to be within the required distance for energy transfer (see Fig. 1). This issue assumes relevance in view of the highly crowded nature of the cell membrane (3Takamori S. Holt M. Jahn R. et al.Molecular anatomy of a trafficking organelle.Cell. 2006; 127: 831-846Abstract Full Text Full Text PDF PubMed Scopus (1679) Google Scholar). FRET from such noninteracting (bystander) pairs complicates the interpretation of FRET results. To our knowledge, King et al. (2King C. Sarabipour S. Hristova K. et al.The FRET signatures of non-interacting proteins in membranes: simulations and experiments.Biophys. J. 2014; 106: 1309-1317Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) have offered, for the first time, an experimentally verified theoretical framework for membrane proteins, which can be effectively used to correct for bystander FRET. FRET is a powerful biophysical tool for determining proximity relationships between fluorescently-tagged macromolecules. The photophysical consequences of FRET from an initially excited donor molecule to an acceptor molecule are well understood, and include 1), the quenching of donor emission and donor excited state lifetimes and 2), the increase in sensitized emission from the acceptor and the corresponding kinetics of the sensitized emission. These changes in photophysics can be quantitatively converted into an energy transfer efficiency that is related to the proximity between donor and acceptor probes on the 1–10 nm scale (4Piston D.W. Kremers G.-J. Fluorescent protein FRET: the good, the bad and the ugly.Trends Biochem. Sci. 2007; 32: 407-414Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar). Once an energy transfer efficiency is extracted experimentally, one is faced with the problem of how to interpret the experimental results. For dilute complexes in solution, there are multiple factors that affect measured energy transfer efficiencies. These are:1.Spectral overlap between donor emission and acceptor absorption,2.The orientation between donor and acceptor transition moment dipoles,3.Stoichiometry,4.Proportion of fluorophores as free and bound to the complex, and5.Distance between the donor and acceptor.For well-characterized systems in solution, some of these factors can be taken into account and reasonable estimates of distances, or indeed relative changes in distance, can be extracted. For membrane proteins, where cellular expression systems could lead to high levels of proteins at the cell membrane due to lack of control in the expression levels (5Meyer B.H. Segura J.-M. Vogel H. et al.FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells.Proc. Natl. Acad. Sci. USA. 2006; 103: 2138-2143Crossref PubMed Scopus (188) Google Scholar), the possibility of FRET occurring from proximal but noninteracting molecules needs to be taken into account (Fig. 1). This is crucial for interpreting FRET in membranes in terms of protein-protein interactions or oligomeric state of membrane proteins. Energy transfer between randomly distributed donors and acceptors in a two-dimensional plane (such as the biological membrane) has been the subject of many theoretical and experimental studies (6Fung B.K. Stryer L. Surface density determination in membranes by fluorescence energy transfer.Biochemistry. 1978; 17: 5241-5248Crossref PubMed Scopus (359) Google Scholar, 7Wolber P.K. Hudson B.S. An analytic solution to the Förster energy transfer problem in two dimensions.Biophys. J. 1979; 28: 197-210Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 8Dewey T.G. Hammes G.G. Calculation on fluorescence resonance energy transfer on surfaces.Biophys. J. 1980; 32: 1023-1035Abstract Full Text PDF PubMed Scopus (150) Google Scholar, 9Snyder B. Freire E. Fluorescence energy transfer in two dimensions. A numeric solution for random and nonrandom distributions.Biophys. J. 1982; 40: 137-148Abstract Full Text PDF PubMed Scopus (101) Google Scholar). However, until now there has been no reliable experimental system for membrane proteins to test the theoretical predictions of these models. The article by King et al. (2King C. Sarabipour S. Hristova K. et al.The FRET signatures of non-interacting proteins in membranes: simulations and experiments.Biophys. J. 2014; 106: 1309-1317Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) addresses an important issue in the usage of FRET to determine quaternary structures of membrane proteins. What is the contribution of bystander or proximity to the measured FRET efficiency? The authors achieve this goal through two methods. First, they use simulations of model oligomeric distributions to extract theoretical proximity FRET values as a function of acceptor concentration. The novelty here is the effect of oligomeric state (i.e., dimer, trimer, or tetramer) on proximity FRET, which has not been examined previously. Second, the authors use YFP donor/mCherry acceptor monomeric membrane protein constructs as experimental model systems for examining proximity FRET. The experimental results agree well with the theoretical framework, even allowing determination of distances of closest approach. The implications for experimental design in future FRET experiments are clear. Expression levels should be kept to a minimum to avoid bystander FRET. According to the experiments of King et al. (2King C. Sarabipour S. Hristova K. et al.The FRET signatures of non-interacting proteins in membranes: simulations and experiments.Biophys. J. 2014; 106: 1309-1317Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), a 20% bystander FRET efficiency corresponds to an acceptor density of 2000 molecules/μm2. Given that typical cell surface areas range between 1000 and 5000 μm2, this density corresponds to an expression level of 2–10 × 106 proteins per cell. Such high levels of expression would lead to complications due to bystander FRET and should be avoided. This article makes a significant contribution to understanding the limitations of FRET-based approaches to membrane protein structure determination, and could serve as a benchmark for exploring organization and interactions of membrane proteins utilizing FRET. We thank G. Aditya Kumar for help with the figure. The FRET Signatures of Noninteracting Proteins in Membranes: Simulations and ExperimentsKing et al.Biophysical JournalMarch 18, 2014In BriefFörster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence-specific interactions, these experiments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of ∼100 Å. This effect does not reflect specific interactions in the membrane and is frequently unappreciated, despite the fact that its magnitude can be significant. Full-Text PDF Open Archive