Diffusion behavior of the fluorescent proteins eGFP and Dreiklang in solvents of different viscosity monitored by fluorescence correlation spectroscopy

Abstract

Abstract:Fluorescence correlation spectroscopy relies on temporal autocorrelation analysis of fluorescence intensity fluctuations that spontaneously arise in systems at equilibrium due to molecular motion and changes of state that cause changes in fluorescence, such as triplet state transition, photoisomerization and other photophysical transformations, to determine the rates of these processes. The stability of a fluorescent molecule against dark state conversion is of particular concern for chromophores intended to be used as reference tags for comparing diffusion processes on multiple time scales. In this work, we analyzed properties of two fluorescent proteins, the photoswitchable Dreiklang and its parental eGFP, in solvents of different viscosity to vary the diffusion time through the observation volume element by several orders of magnitude. In contrast to eGFP, Dreiklang undergoes a dark-state conversion on the time scale of tens to hundreds of microseconds under conditions of intense fluorescence excitation, which results in artificially shortened diffusion times if the diffusional motion through the observation volume is sufficiently slowed down. Such photophysical quenching processes have also been observed in FCS studies on other photoswitchable fluorescent proteins including Citrine, from which Dreiklang was derived by genetic engineering. This property readily explains the discrepancies observed previously between the diffusion times of eGFP-and Dreiklang-labeled plasma membrane protein complexes.

Media Modulation based Molecular Communication

“…(t, z RX , z TX )dz RX (t, z RX , z TX )f z TX (z TX ) dz RX dz TX b − z TX − vt √ 4D t − erf z RX a − z TX − vt √ 4D t dz TX ,(39)where E{•} denotes the expectation operator, and we exploit in (a) the uniform distribution of z TX , i.e., f z TX (z TX ) = 1 l TX , z TX a ≤ z TX ≤ z TX b . Finally, applying the integral erf(x)dx = x erf(x) + 1 √ π exp (−x 2 ), we obtain(21). This concludes the proof.F.…”

A Method to Quantify Molecular Diffusion within Thin Solvated Polymer Films: A Case Study on Films of Natively Unfolded Nucleoporins

“…Mean and standard deviations for 8 data points are shown (2 data points per image, left and right of the center, from a total of 4 images selected from 2 independent measurements); mobile fraction and bleaching parameter across these measurements were roughly constant: k = 0.84 ± 0.02 and K 0 = 0.93 ± 0.05. (E) Comparison, for GFP NTR and GFP Std , of diffusion constants in bulk solution (“no Nsp1”; taken from ref ( 38 )) and for FG Nsp1 concentrations of approximately 100 and 500 mg/mL, corresponding to a virtually uncompressed (30 nm thick) and strongly compressed (6 nm thick) FG Nsp1 film, respectively.…”

“…From analogous measurements with GFP Std (Supporting Figure S5) we estimate D = 6.5 ± 1.9 μm 2 /s for the unperturbed FG domain film. For comparison, the diffusion coefficient of GFP in aqueous solution has been determined by fluorescence correlation spectroscopy to be D = 90 ± 3 μm 2 / s. 38 Thus, the FG Nsp1 film reduces the diffusion of GFP Std (and likely also GFP Inert ) by about an order of magnitude, while GFP NTR experiences a further reduction by a moderate few fold.…”

“…(E) Comparison, for GFP NTR and GFP Std , of diffusion constants in bulk solution ('no FG Nsp1 '; taken from ref. 24) and for FG Nsp1 concentrations of approximately 100 and 500 mg/mL, corresponding to a virtually uncompressed (30 nm thick) and strongly compressed (6 nm thick) FG Nsp1 film, respectively.…”