Native and Unfolded States of Phosphoglycerate Kinase Studied by Single‐Molecule FRET

Rosenkranz, Tobias; Schlesinger, Ramona; Gabba, Matteo; Fitter, Jörg

doi:10.1002/cphc.201000701

Cited by 20 publications

(25 citation statements)

References 60 publications

(73 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To be sensitive to interdomain distance changes induced by substrate binding, we choose two labeling positions, each at the distal end of the N-and the C-domain, respectively (18,32). For this purpose, we employed a cysteine double mutant of PGK from baker's yeast, which was site-specifically labeled with a donor (Alexa Fluor 488) and an acceptor (Alexa Fluor 647) fluorophore (see Fig.…”

Section: Single-molecule Fret Measurementsmentioning

confidence: 99%

Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

Gabba

Poblete

Rosenkranz

et al. 2014

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.

show abstract

Section: Single-molecule Fret Measurementsmentioning

confidence: 99%

Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

Gabba

Poblete

Rosenkranz

et al. 2014

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…that was used to model the accessible position distributions for the dyes after accounting for linker flexibility53. The software was benchmarked by computing the native state FRET efficiency histogram of phosphoglycerate kinase (RCSB PDB ID: 3PGK) and comparing to experiment54. The parameters used in the simulation (i.e.…”

Section: Methodsmentioning

confidence: 99%

Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY

Shi

Nobrega

Schwantes

et al. 2017

Sci Rep

View full text Add to dashboard Cite

The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. Here, we report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structure of the excited state ensemble. This prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. Using these results, we then predict incisive single molecule FRET experiments as a means of model validation. This study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.

show abstract

“…For a fourth example, we analyzed a conventionally attached dye (Alexa 488 malemide; Figure 4 B) bound to a cysteine residue in the structure of phosphoglycerate kinase (PGK). [25] Details about this construct are given in ref. [25].…”

Section: Fluorescence Anisotropy Decay Of Full-length Flash-cam Complmentioning

confidence: 99%

Nanosecond Dynamics of Calmodulin and Ribosome‐Bound Nascent Chains Studied by Time‐Resolved Fluorescence Anisotropy

et al. 2014

Self Cite

View full text Add to dashboard Cite

We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full-length, half-length, and first EF-hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site-specifically incorporated in the first N-terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome-tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome.

show abstract

Native and Unfolded States of Phosphoglycerate Kinase Studied by Single‐Molecule FRET

Cited by 20 publications

References 60 publications

Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY

Nanosecond Dynamics of Calmodulin and Ribosome‐Bound Nascent Chains Studied by Time‐Resolved Fluorescence Anisotropy

Contact Info

Product

Resources

About