2 research outputs found
Combining Graphical and Analytical Methods with Molecular Simulations To Analyze Time-Resolved FRET Measurements of Labeled Macromolecules Accurately
Förster resonance energy transfer
(FRET) measurements from
a donor, D, to an acceptor, A, fluorophore are frequently used <i>in vitro</i> and in live cells to reveal information on the
structure and dynamics of DA labeled macromolecules. Accurate descriptions
of FRET measurements by molecular models are complicated because the
fluorophores are usually coupled to the macromolecule via flexible
long linkers allowing for diffusional exchange between multiple states
with different fluorescence properties caused by distinct environmental
quenching, dye mobilities, and variable DA distances. It is often
assumed for the analysis of fluorescence intensity decays that DA
distances and D quenching are uncorrelated (homogeneous quenching
by FRET) and that the exchange between distinct fluorophore states
is slow (quasistatic). This allows us to introduce the FRET-induced
donor decay, ε<sub>D</sub>(<i>t</i>), a function solely
depending on the species fraction distribution of the rate constants
of energy transfer by FRET, for a convenient joint analysis of fluorescence
decays of FRET and reference samples by integrated graphical and analytical
procedures. Additionally, we developed a simulation toolkit to model
dye diffusion, fluorescence quenching by the protein surface, and
FRET. A benchmark study with simulated fluorescence decays of 500
protein structures demonstrates that the quasistatic homogeneous model
works very well and recovers for single conformations the average
DA distances with an accuracy of < 2%. For more complex
cases, where proteins adopt multiple conformations with significantly
different dye environments (heterogeneous case), we introduce a general
analysis framework and evaluate its power in resolving heterogeneities
in DA distances. The developed fast simulation methods, relying on
Brownian dynamics of a coarse-grained dye in its sterically accessible
volume, allow us to incorporate structural information in the decay
analysis for heterogeneous cases by relating dye states with protein
conformations to pave the way for fluorescence and FRET-based dynamic
structural biology. Finally, we present theories and simulations to
assess the accuracy and precision of steady-state and time-resolved
FRET measurements in resolving DA distances on the single-molecule
and ensemble level and provide a rigorous framework for estimating
approximation, systematic, and statistical errors
Triphosphate Induced Dimerization of Human Guanylate Binding Protein 1 Involves Association of the C‑Terminal Helices: A Joint Double Electron–Electron Resonance and FRET Study
Human guanylate binding protein 1
(hGBP1) is a member of the dynamin
superfamily of large GTPases. During GTP hydrolysis, the protein undergoes
structural changes leading to self-assembly. Previous studies have
suggested dimerization of the protein by means of its large GTPase
(LG) domain and significant conformational changes in helical regions
near the LG domain and at its C-terminus. We used site-directed labeling
and a combination of pulsed electron paramagnetic resonance and time-resolved
fluorescence spectroscopy for structural investigations on hGBP1 dimerization
and conformational changes of its C-terminal helix α13. Consistent
distance measurements by double electron–electron resonance
(DEER, also named pulse double electron resonance = PELDOR) spectroscopy
and Förster resonance energy transfer (FRET) measurements using
model-free analysis approaches revealed a close interaction of the
two α13 helices in the hGBP1 dimer formed upon binding of the
nonhydrolyzable nucleoside triphosphate derivate GppNHp. In molecular
dynamics (MD) simulations, these two helices form a stable dimer in
solution. Our data show that dimer formation of hGBP1 involves multiple
spatially distant regions of the protein, namely, the N-terminal LG
domain and the C-terminal helices α13. The contacts formed between
the two α13 helices and the resulting juxtaposition are expected
to be a key step for the physiological membrane localization of hGBP1
through the farnesyl groups attached to the end of α13