Abstract
Green fluorescent protein (GFP) mutants have become the most widely used fluorescence markers in the life sciences, and although they are becoming increasingly popular as mechanical force or strain probes, there is little direct information on how their fluorescence changes when mechanically stretched. Here we derive high-resolution structural models of the mechanical intermediate states of stretched GFP using steered molecular dynamics (SMD) simulations. These structures were used to produce mutants of EGFP and EYFP that mimic GFP's different mechanical intermediates. A spectroscopic analysis revealed that a population of EGFP molecules with a missing N-terminal α-helix was significantly dimmed, while the fluorescence lifetime characteristic of the anionic chromophore state remained unaffected. This suggests a mechanism how N-terminal deletions can switch the protonation state of the chromophore, and how the fluorescence of GFP molecules in response to mechanical disturbance might be turned off.
Highlights
Since the discovery that Green fluorescent protein (GFP) can be used to monitor gene expression and protein localization in living organisms [1], GFP and its mutants have become the most widely used fluorescence markers to identify how protein expression levels and their spatial organization within cells and tissues are altered
There have been a number of AFM studies of GFP [11,12,13,14,15], and computational simulations to analyze how GFP might unfold under tensile forces, including a self-organized polymer models [14,16], a coarse-grained elastic network model [17], and an Ising-like model [18], we present here the first atomic-level structural models of the forced unfolding trajectories of GFP as derived from steered molecular dynamics (SMD) simulations
Several plateau regions exist in the extension-time plots (Figure 1) indicating the existence of multiple mechanical intermediate states separated by energy barriers that have to be passed before further unfolding can occur
Summary
Since the discovery that GFP can be used to monitor gene expression and protein localization in living organisms [1], GFP and its mutants have become the most widely used fluorescence markers to identify how protein expression levels and their spatial organization within cells and tissues are altered. When mapping spatial distributions of GFP-tagged protein within cells, the lack of GFP fluorescence is typically interpreted as an absence of the tagged protein in that region. If GFP constructs are used as mechanical sensors, it is prudent to consider how stretching GFP derivatives might impact their fluorescence. To further study the putative spectroscopic properties of the mechanical intermediates where terminal peptide sequences were removed, and in analogy to previous approaches [19], we used mutants of EGFP and EYFP designed to mimic the structures of the mechanical intermediate states seen in SMD simulations
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