Estimating the distance separating fluorescent protein FRET pairs

Abstract

Förster resonance energy transfer (FRET) describes a physical phenomenon widely applied in biomedical research to estimate separations between biological molecules. Routinely, genetic engineering is used to incorporate spectral variants of the green fluorescent protein (GFPs), into cellular expressed proteins. The transfer efficiency or rate of energy transfer between donor and acceptor FPs is then assayed. As appreciable FRET occurs only when donors and acceptors are in close proximity (1–10 nm), the presence of FRET may indicate that the engineered proteins associate as interacting species. For a homogeneous population of FRET pairs the separations between FRET donors and acceptors can be estimated from a measured FRET efficiency if it is assumed that donors and acceptors are randomly oriented and rotate extensively during their excited state (dynamic regime). Unlike typical organic fluorophores, the rotational correlation-times of FPs are typically much longer than their fluorescence lifetime; accordingly FPs are virtually static during their excited state. Thus, estimating separations between FP FRET pairs is problematic. To overcome this obstacle, we present here a simple method for estimating separations between FPs using the experimentally measured average FRET efficiency. This approach assumes that donor and acceptor fluorophores are randomly oriented, but do not rotate during their excited state (static regime). This approach utilizes a Monte-Carlo simulation generated look-up table that allows one to estimate the separation, normalized to the Förster distance, from the average FRET efficiency. Assuming a dynamic regime overestimates the separation significantly (by 10% near 0.5 and 30% near 0.75 efficiencies) compared to assuming a static regime, which is more appropriate for estimates of separations between FPs.

Covert Changes in CaMKII Holoenzyme Structure Identified for Activation and Subsequent Interactions

“…3 B). The FRET efficiencies of two structurally related hetero-FRET standards, C17V and C32V indicate that the difference in separation for these constructs corresponds to a change of~4 Å (54). We conclude that these changes reflect subtle and previously covert transformations in the nature of catalytic-domain pairing in response to Ca 2þ /CaM binding and subsequent T286 autophosphorylation.…”

“…3 E). Care must be exercised when interpreting FRET changes (50)(51)(52), as a reduction might reflect a change in fluorophore orientation rather than proximity (53,54). Because Venus was attached to the catalyticdomain via a flexible 15 amino-acid linker in all CaMKII constructs used in this study, these changes most likely reflect an increase in separation.…”

Tunable molecular tension sensors reveal extension-based control of vinculin loading

“…, the physical separation of the organic dyes (E), and the relative orientation and timescale of rotation of the dyes (F). Note when modeling large FP-based TSMods, the static isotropic regime of energy transfer is valid, while FRET between organic dyes is more appropriately described by the dynamic isotropic assumption of the classical Förster equation (Vogel et al, 2014). Figure 2-Figure supplement 1A).…”

Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy

“…The eGFP tag was selected as a FRET donor, due to its high quantum yield (0.6) and its monoexponential time-resolved fluorescence decay. The mCherry tag was used as an acceptor due to the strong overlap between its absorption spectrum and the eGFP emission spectrum, resulting in a large Förster distance, R 0 (about 54 Å) [62,69,100]. The labeled Vpr proteins were found to accumulate mainly at the nuclear envelope, though some proteins were also expressed in the cytoplasm and the nucleus.…”