Quantitative Understanding of the Energy Transfer between Fluorescent Proteins Connected via Flexible Peptide Linkers

Abstract

The fusion of different protein domains via peptide linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser linkers are discussed.

Dynamics simulations for engineering macromolecular interactions

“…Evers et al 41 previously determined FRET signals as a function of the linker length between these proteins, and obtained similar results to our predictions, which supports our approach. One factor in the F€ orster theory of FRET is j, which is a function of the angles between the dipoles in the fluorescent proteins.…”

“…5. Calculated energy-transfer efficiency between ECFP and EYFP joined by a flexible glycine-serine linker as described in Evers et al 41 The energytransfer efficiency was calculated according to the formula for F€ orster distance which depends on j 2 , which is in turn a function of the relative position and orientation of the fluorophores. We simulated ECFP-linker-EYFP configurations with varying linker lengths and calculated the energy-transfer efficiency with both (a) per-frame calculated j 2 and (b) constant j 2 ¼ 2=3.…”

“…Evers et al 41 performed FRET studies to determine how different lengths of a flexible glycine-serine linker affect interdomain distances. They used a simple modeling approach treating the linker as a random coil structure in order to examine the behavior of their systems.…”

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…The lifetime Table 3 distribution of the EGFP-(L) 6 -mCherry construct has a pattern of three lifetimes at 2.49, 1.18 and 0.35 ns with amplitudes listed in Table 3. The short linker of six amino acids (GSGSGS) puts a constraint of relatively small distances between both fluorescent proteins (Evers et al 2006). Because the linker peptide has a very flexible structure, it is very possible that we are facing a distribution of relative distances and orientations between both transition moments.…”

Metal‐Induced Specific and Nonspecific Oligonucleotide Folding Studied by FRET and Related Biophysical and Bioanalytical Implications

“…It is likely that the DNA has a distribution of end-to-end distance due to its non-rigid structure. [43] Figure 5. The calculated FRET ratio (left axis) and FRET efficiency (right axis) as a function of endto-end distance change for the Hg II FRET probe.…”