10 research outputs found
The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins
International audienceUnderstanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.</p
The Mechanochemistry of a Structural Zinc Finger
Zinc fingers are highly ubiquitous
structural motifs that provide
stability to proteins, thus contributing to their correct folding.
Despite the high thermodynamic stability of the ZnCys<sub>4</sub> centers,
their kinetic properties display remarkable lability. Here, we use
a combination of protein engineering with single molecule force spectroscopy
atomic force microscopy (AFM) to uncover the surprising mechanical
lability (∼90 pN) of the individual Zn–S bonds that
form the two equivalent zinc finger motifs embedded in the structure
of the multidomain DnaJ chaperone. Rational mutations within the zinc
coordinating residues enable direct identification of the chemical
determinants that regulate the interplay between zinc bindingrequiring
the presence of all four cysteinesand disulfide bond formation.
Finally, our observations show that binding to hydrophobic short peptides
drastically increases the mechanical stability of DnaJ. Altogether,
our experimental approach offers a detailed, atomistic vista on the
fine chemical mechanisms that govern the nanomechanics of individual,
naturally occurring zinc finger
DNA Binding Induces a Nanomechanical Switch in the RRM1 Domain of TDP-43
Understanding the molecular mechanisms governing protein-nucleic acid interactions is fundamental to many nuclear processes. However, how nucleic acid binding affects the conformation and dynamics of the substrate protein remains poorly understood. Here we use a combination of single molecule force spectroscopy AFM and biochemical assays to show that the binding of TG-rich ssDNA triggers a mechanical switch in the RRM1 domain of TDP-43, toggling between an entropic spring devoid of mechanical stability and a shock absorber bound-form that resists unfolding forces of ∼40 pN. The fraction of mechanically resistant proteins correlates with an increasing length of the TGn oligonucleotide, demonstrating that protein mechanical stability is a direct reporter of nucleic acid binding. Steered molecular dynamics simulations on related RNA oligonucleotides reveal that the increased mechanical stability fingerprinting the holo-form is likely to stem from a unique scenario whereby the nucleic acid acts as a 'mechanical staple' that protects RRM1 from mechanical unfolding. Our approach highlights nucleic acid binding as an effective strategy to control protein nanomechanics
Single-molecule force spectroscopy predicts a misfolded, domain-swapped conformation in human γD-crystallin protein
Cataract is a protein misfolding disease where the size of the aggregate is directly related to the severity of the disorder. However, the molecular mechanisms that trigger the onset of aggregation remain unknown. Here we use a combination of protein engineering techniques and single-molecule force spectroscopy using atomic force microscopy to study the individual unfolding pathways of the human γD-crystallin, a multidomain protein that must remain correctly folded during the entire lifetime to guarantee lens transparency. When stretching individual polyproteins containing two neighboring HγD-crystallin monomers, we captured an anomalous misfolded conformation in which the β1 and β2 strands of the N terminus domain of two adjacent monomers swap. This experimentally elusive domain-swapped conformation is likely to be responsible for the increase in molecular aggregation that we measure in vitro. Our results demonstrate the power of force spectroscopy at capturing rare misfolded conformations with potential implications for the understanding of the molecular onset of protein aggregation
DNA binding induces a nanomechanical switch in the RRM1 domain of TDP-43
Understanding the molecular mechanisms governing protein-nucleic acid interactions is fundamental to many nuclear processes. However, how nucleic acid binding affects the conformation and dynamics of the substrate protein remains poorly understood. Here we use a combination of single molecule force spectroscopy AFM and biochemical assays to show that the binding of TG-rich ssDNA triggers a mechanical switch in the RRM1 domain of TDP-43, toggling between an entropic spring devoid of mechanical stability and a shock absorber bound-form that resists unfolding forces of ∼40 pN. The fraction of mechanically resistant proteins correlates with an increasing length of the TGn oligonucleotide, demonstrating that protein mechanical stability is a direct reporter of nucleic acid binding. Steered molecular dynamics simulations on related RNA oligonucleotides reveal that the increased mechanical stability fingerprinting the holo-form is likely to stem from a unique scenario whereby the nucleic acid acts as a 'mechanical staple' that protects RRM1 from mechanical unfolding. Our approach highlights nucleic acid binding as an effective strategy to control protein nanomechanics
The mechanical stability of proteins regulates their translocation rate into the cell nucleus
DNA Binding Induces a Nanomechanical Switch in the RRM1 Domain of TDP-43
Understanding
the molecular mechanisms governing protein–nucleic
acid interactions is fundamental to many nuclear processes. However,
how nucleic acid binding affects the conformation and dynamics of
the substrate protein remains poorly understood. Here we use a combination
of single molecule force spectroscopy AFM and biochemical assays to
show that the binding of TG-rich ssDNA triggers a mechanical switch
in the RRM1 domain of TDP-43, toggling between an entropic spring
devoid of mechanical stability and a shock absorber bound-form that
resists unfolding forces of ∼40 pN. The fraction of mechanically
resistant proteins correlates with an increasing length of the TG<sub><i>n</i></sub> oligonucleotide, demonstrating that protein
mechanical stability is a direct reporter of nucleic acid binding.
Steered molecular dynamics simulations on related RNA oligonucleotides
reveal that the increased mechanical stability fingerprinting the
holo-form is likely to stem from a unique scenario whereby the nucleic
acid acts as a “mechanical staple” that protects RRM1
from mechanical unfolding. Our approach highlights nucleic acid binding
as an effective strategy to control protein nanomechanics
DNA Binding Induces a Nanomechanical Switch in the RRM1 Domain of TDP-43
Understanding
the molecular mechanisms governing protein–nucleic
acid interactions is fundamental to many nuclear processes. However,
how nucleic acid binding affects the conformation and dynamics of
the substrate protein remains poorly understood. Here we use a combination
of single molecule force spectroscopy AFM and biochemical assays to
show that the binding of TG-rich ssDNA triggers a mechanical switch
in the RRM1 domain of TDP-43, toggling between an entropic spring
devoid of mechanical stability and a shock absorber bound-form that
resists unfolding forces of ∼40 pN. The fraction of mechanically
resistant proteins correlates with an increasing length of the TG<sub><i>n</i></sub> oligonucleotide, demonstrating that protein
mechanical stability is a direct reporter of nucleic acid binding.
Steered molecular dynamics simulations on related RNA oligonucleotides
reveal that the increased mechanical stability fingerprinting the
holo-form is likely to stem from a unique scenario whereby the nucleic
acid acts as a “mechanical staple” that protects RRM1
from mechanical unfolding. Our approach highlights nucleic acid binding
as an effective strategy to control protein nanomechanics