Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells

PLOS ONE RESEARCH ARTICLE Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells Annett Petrich1, Amit Koikkarah Aji1, Valentin Dunsing1,2, Salvatore Chiantia ID1* 1 University of Potsdam, Institute of Biochemistry and Biology, Potsdam, Germany, 2 Aix-Marseille University, CNRS, UMR 7288, IBDM, Turing Center for Living Systems, Marseille, France a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Petrich A, Aji AK, Dunsing V, Chiantia S (2023) Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells. PLoS ONE 18(8): e0285486. https://doi.org/10.1371/journal. pone.0285486 Editor: Rajiv Kumar Kar, Indian Institute of Technology Guwahati, INDIA Received: February 15, 2023 Accepted: April 25, 2023 Published: August 3, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0285486 Copyright: © 2023 Petrich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All plot data are included in this online submission as Excel file. Funding: Deutsche Forschungsgemeinschaft (DFG) project number 407961559 to S.C. www. * Abstract Protein-protein-interactions play an important role in many cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced “blinking” and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N&B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for protein-protein-interactions quantification via fluorescence fluctuation-based methods. Introduction A multitude of cellular processes, such as biomolecule transport, ion channel activity, cell-cell adhesion and communication are regulated by protein-protein-interactions (PPIs) [1–3]. “Classical” bulk biochemical in vitro methods that are used to quantify PPIs (e.g., co- PLOS ONE | https://doi.org/10.1371/journal.pone.0285486 August 3, 2023 1 / 13 PLOS ONE Benchmarking of green fluorescent proteins via sFCS and N&B dfg.de HFSP long-term postdoctoral fellowship (HFSP LT0058/2022-L) to V.D. www.hsfp.org The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: FFS, fluorescence fluctuation spectroscopy; FP, fluorescent protein; N&B, number and brightness; pf, fluorescence probability; PM, plasma membrane; PPI, proteinprotein-interaction; sFCS, scanning fluorescence correlation spectroscopy. immunoprecipitation (co-IP), pull-down assays and western blotting) cannot be used to obtain information about intracellular protein distribution in live-cell samples or to monitor the effects of variations in concentrations between different cells [4, 5]. Conventional optical microscopy can visualize the localization of proteins, but its resolution is limited [4, 6]. More complex approaches, such as fluorescence fluctuation spectroscopy (FFS), can assess the interactions between molecules in complexes and obtain insights into cellular pathways and assembly processes [4–8]. FFS provides information about dynamics through the analysis of signal fluctuations from fluorescently labeled molecules [6, 7, 9]. Additionally, the magnitude of such fluctuations can be used to derive quantitative information about the multimerization state (i.e., number of monomers in a multimer) of the protein of interest [7–10]. A common strategy to investigate PPIs in cellula via FFS is the fusion of a fluorescent protein (FP) to the protein of interest [7–9, 11]. By comparing the brightness of protein multimers tagged with FPs to the brightness of a monomeric reference, it is possible to quantify the number of FP monomers in the complex and, thus, the oligomerization state of the protein of interest [5, 7, 12]. A major problem for several FFS applications that rely on FPs, though, is the presence of non-emitting “dark” proteins, which can be quantified through the so-called fluorescence probability (pf) [7, 13]. It is sometime assumed that FPs have a pf value of 1 meaning that, for example, a trimer containing three FPs emits in average a three-fold higher signal than a monomer labelled with one FP [5, 7]. Instead, pf values of e.g. green emitting FPs have been reported in the range between 0.5 and 0.8 [1, 2, 7, 13–19]. Recently, we have systematically quantified the pf of several FPs, focusing mainly on proteins emitting in the red part of the visible spectrum [7]. In summary, since high pf values are required for increased sensitivity, not all FPs are equally suitable for oligomerization studies [7]. Because of the presence of nonemitting FPs (i.e., pf lower than 1), FFS approaches might underestimate the amount of FPs and therefore, the oligomeric state of the protein of interest. Some of the most used FPs are the green fluorescent protein (GFP) and its mutants, such as the monomer (...truncated)