In-cell single-molecule FRET measurements reveal three conformational state changes in RAF protein.
Alternative laser excitation (ALEX)
CRAF
Förster resonance energy transfer (FRET)
In-cell measurement
Protein structure
Single molecule
Journal
Biochimica et biophysica acta. General subjects
ISSN: 1872-8006
Titre abrégé: Biochim Biophys Acta Gen Subj
Pays: Netherlands
ID NLM: 101731726
Informations de publication
Date de publication:
02 2020
02 2020
Historique:
received:
29
12
2018
revised:
26
04
2019
accepted:
30
04
2019
pubmed:
10
5
2019
medline:
14
7
2020
entrez:
10
5
2019
Statut:
ppublish
Résumé
The structures of proteins are intimately related to their functions. Significant efforts have been dedicated to the structural investigation of proteins, mainly those of purified proteins in in vitro environments. Proteins function in living cells and thus protein structures must be regulated by interactions with various molecules, some of which participate in reaction networks, depending on the states, conditions, or actions of the cell. Therefore, it is very important to understand the structural behavior of proteins in living cells. Single-molecule Förster resonance energy transfer (smFRET) measurements were conducted using the alternative laser excitation (ALEX) technique. smFRET distributions of cytosolic Rapidly Accelerated Fibrosarcoma (RAF) proteins in living HeLa cells were obtained with exclusion of the negative effects of photobleached fluorophores and incompletely labeled proteins on smFRET. smFRET histograms of wildtype (wt) RAF in live cells exhibited two major peaks, whereas that of the S621A mutant, which has been thought to have an expanded structure, was almost single-peaked. A population shift involving the peaks for wt RAF was detected upon epidermal growth factor stimulation. Spontaneous transitions between the conformational states corresponding to the two peaks were also detected using the FRET-two-channel kernel-based density distribution estimator method in comparison to static double-stranded DNA samples. Cytosolic CRAF has at least three conformational states; in addition to the closed and open forms, the fully-open form was distinctly specified. Based on the results, we propose a speculative structural model for CRAF. Structural distribution and changes to proteins in live cells as a result of intracellular interactions were successfully identified. smFRET using ALEX is applicable to any other cytosolic proteins.
Sections du résumé
BACKGROUND
The structures of proteins are intimately related to their functions. Significant efforts have been dedicated to the structural investigation of proteins, mainly those of purified proteins in in vitro environments. Proteins function in living cells and thus protein structures must be regulated by interactions with various molecules, some of which participate in reaction networks, depending on the states, conditions, or actions of the cell. Therefore, it is very important to understand the structural behavior of proteins in living cells.
METHODS
Single-molecule Förster resonance energy transfer (smFRET) measurements were conducted using the alternative laser excitation (ALEX) technique. smFRET distributions of cytosolic Rapidly Accelerated Fibrosarcoma (RAF) proteins in living HeLa cells were obtained with exclusion of the negative effects of photobleached fluorophores and incompletely labeled proteins on smFRET.
RESULTS
smFRET histograms of wildtype (wt) RAF in live cells exhibited two major peaks, whereas that of the S621A mutant, which has been thought to have an expanded structure, was almost single-peaked. A population shift involving the peaks for wt RAF was detected upon epidermal growth factor stimulation. Spontaneous transitions between the conformational states corresponding to the two peaks were also detected using the FRET-two-channel kernel-based density distribution estimator method in comparison to static double-stranded DNA samples.
CONCLUSIONS
Cytosolic CRAF has at least three conformational states; in addition to the closed and open forms, the fully-open form was distinctly specified. Based on the results, we propose a speculative structural model for CRAF.
GENERAL SIGNIFICANCE
Structural distribution and changes to proteins in live cells as a result of intracellular interactions were successfully identified. smFRET using ALEX is applicable to any other cytosolic proteins.
Identifiants
pubmed: 31071411
pii: S0304-4165(19)30114-X
doi: 10.1016/j.bbagen.2019.04.022
pii:
doi:
Substances chimiques
Fluorescent Dyes
0
Epidermal Growth Factor
62229-50-9
Proto-Oncogene Proteins c-raf
EC 2.7.11.1
Raf1 protein, human
EC 2.7.11.1
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
129358Informations de copyright
Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.