Limits...
Force-induced remodelling of proteins and their complexes.

Chen Y, Radford SE, Brockwell DJ - Curr. Opin. Struct. Biol. (2015)

Bottom Line: The effects of force on the biophysical properties of biological systems can be large and varied.As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results.Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

No MeSH data available.


Related in: MedlinePlus

The highly avid E9:Im9 complex is a force-sensitive trip bond [73]. (a) When extended between residue 3 of E9 (green) and 81 of Im9 (pink), the complex dissociates at low force with a short lifetime (12.5 ms under 20 pN force) due to force-induced remodelling of the binding interface which is connected directly to the N-terminus of E9. (See left hand inset showing simplified topology diagram for the complex. Yellow filled circles designate pulling points). Interface remodelling can be prevented by introducing a disulfide cross-link (between residues 20 and 66, highlighted in blue and blue circles in the structure and topology diagrams, respectively), diverting the force propagation network away from regions proximal to the interface. This results in a mechanically strong, long lived complex (3.9 hours under 20 pN force), with a dissociation rate constant identical to that measured by ensemble methods. If a disulphide bond is introduced between residues 31 and 122 of E9 (red and red circles in the structure and topology diagrams, respectively), remodelling is not prevented, and the complex behaves identically to the wild-type (WT) protein. (b) Dynamic force spectrum of the wild-type complex (black circles), E9 cross-linked between residues 20–66 (blue closed circles) and 31–122 (red circles).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4499843&req=5

Figure 4: The highly avid E9:Im9 complex is a force-sensitive trip bond [73]. (a) When extended between residue 3 of E9 (green) and 81 of Im9 (pink), the complex dissociates at low force with a short lifetime (12.5 ms under 20 pN force) due to force-induced remodelling of the binding interface which is connected directly to the N-terminus of E9. (See left hand inset showing simplified topology diagram for the complex. Yellow filled circles designate pulling points). Interface remodelling can be prevented by introducing a disulfide cross-link (between residues 20 and 66, highlighted in blue and blue circles in the structure and topology diagrams, respectively), diverting the force propagation network away from regions proximal to the interface. This results in a mechanically strong, long lived complex (3.9 hours under 20 pN force), with a dissociation rate constant identical to that measured by ensemble methods. If a disulphide bond is introduced between residues 31 and 122 of E9 (red and red circles in the structure and topology diagrams, respectively), remodelling is not prevented, and the complex behaves identically to the wild-type (WT) protein. (b) Dynamic force spectrum of the wild-type complex (black circles), E9 cross-linked between residues 20–66 (blue closed circles) and 31–122 (red circles).

Mentions: While protein topology governs the molecular response to extension to a large degree, the stability of the mechanical interface (the parts of a protein that resist the applied extension) can also affect protein mechanical strength and the degree of co-operativity upon unfolding. For example, molecular dynamics simulations demonstrated that protein L (a protein that is expressed on the outer cell wall of some bacteria but has no known mechanical function) unfolds by the shearing of two mechanical subdomains with an interface between neighbouring anti-parallel N- and C-terminal β-strands [60]. Increasing the hydrophobic contacts (or inter-digitation of side-chains) across this interface increased the mechanical strength of protein L from 134 to 206 pN [61]. In vitro unfolding studies on simple protein polymers have also shown that the mechanical strength of a protein depends on the direction of force application relative to the topology of the secondary structure. A protein may thus be able to resist mechanical deformation when force is applied in one geometry, but be weak when force is applied in another direction (similar to pulling apart Velcro). The anisotropic response of proteins and their complexes to force has now been demonstrated many times [62–73]. These effects, which to a large part were delineated using engineered model poly-proteins unfolded by AFM in vitro, have led to the realisation that proteins and their complexes may exploit different unfolding pathways in the presence and absence of force, leading to force-catalysed or force-triggered phenomena in vivo [36,73]. For example, we have shown that mechanical perturbation remodels the interface of an exceedingly stable complex (Kd = 10−14 M, koff = 1.8 × 10−6 s−1) formed between the bacterial antibiotic nuclease colicin E9 and its inhibitor, immunity protein 9 (Im9) so that dissociation occurs at a surprisingly low force (<20 pN) [73]. Examination of the N-terminal sequence of E9 (through which force or remodelling is applied or carried out in vivo) showed that this region docks against the remainder of the globular domain with little side-chain inter-digitation (see Figure 4). As described for protein L, this is ideal for transmitting mechanical signals to the binding interface at low force. Remodelling increases the off-rate a million-fold relative to that expected for a slip bond, allowing Im9 release and E9 activation at a biologically relevant rate upon binding to a competing organism.


Force-induced remodelling of proteins and their complexes.

Chen Y, Radford SE, Brockwell DJ - Curr. Opin. Struct. Biol. (2015)

The highly avid E9:Im9 complex is a force-sensitive trip bond [73]. (a) When extended between residue 3 of E9 (green) and 81 of Im9 (pink), the complex dissociates at low force with a short lifetime (12.5 ms under 20 pN force) due to force-induced remodelling of the binding interface which is connected directly to the N-terminus of E9. (See left hand inset showing simplified topology diagram for the complex. Yellow filled circles designate pulling points). Interface remodelling can be prevented by introducing a disulfide cross-link (between residues 20 and 66, highlighted in blue and blue circles in the structure and topology diagrams, respectively), diverting the force propagation network away from regions proximal to the interface. This results in a mechanically strong, long lived complex (3.9 hours under 20 pN force), with a dissociation rate constant identical to that measured by ensemble methods. If a disulphide bond is introduced between residues 31 and 122 of E9 (red and red circles in the structure and topology diagrams, respectively), remodelling is not prevented, and the complex behaves identically to the wild-type (WT) protein. (b) Dynamic force spectrum of the wild-type complex (black circles), E9 cross-linked between residues 20–66 (blue closed circles) and 31–122 (red circles).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499843&req=5

Figure 4: The highly avid E9:Im9 complex is a force-sensitive trip bond [73]. (a) When extended between residue 3 of E9 (green) and 81 of Im9 (pink), the complex dissociates at low force with a short lifetime (12.5 ms under 20 pN force) due to force-induced remodelling of the binding interface which is connected directly to the N-terminus of E9. (See left hand inset showing simplified topology diagram for the complex. Yellow filled circles designate pulling points). Interface remodelling can be prevented by introducing a disulfide cross-link (between residues 20 and 66, highlighted in blue and blue circles in the structure and topology diagrams, respectively), diverting the force propagation network away from regions proximal to the interface. This results in a mechanically strong, long lived complex (3.9 hours under 20 pN force), with a dissociation rate constant identical to that measured by ensemble methods. If a disulphide bond is introduced between residues 31 and 122 of E9 (red and red circles in the structure and topology diagrams, respectively), remodelling is not prevented, and the complex behaves identically to the wild-type (WT) protein. (b) Dynamic force spectrum of the wild-type complex (black circles), E9 cross-linked between residues 20–66 (blue closed circles) and 31–122 (red circles).
Mentions: While protein topology governs the molecular response to extension to a large degree, the stability of the mechanical interface (the parts of a protein that resist the applied extension) can also affect protein mechanical strength and the degree of co-operativity upon unfolding. For example, molecular dynamics simulations demonstrated that protein L (a protein that is expressed on the outer cell wall of some bacteria but has no known mechanical function) unfolds by the shearing of two mechanical subdomains with an interface between neighbouring anti-parallel N- and C-terminal β-strands [60]. Increasing the hydrophobic contacts (or inter-digitation of side-chains) across this interface increased the mechanical strength of protein L from 134 to 206 pN [61]. In vitro unfolding studies on simple protein polymers have also shown that the mechanical strength of a protein depends on the direction of force application relative to the topology of the secondary structure. A protein may thus be able to resist mechanical deformation when force is applied in one geometry, but be weak when force is applied in another direction (similar to pulling apart Velcro). The anisotropic response of proteins and their complexes to force has now been demonstrated many times [62–73]. These effects, which to a large part were delineated using engineered model poly-proteins unfolded by AFM in vitro, have led to the realisation that proteins and their complexes may exploit different unfolding pathways in the presence and absence of force, leading to force-catalysed or force-triggered phenomena in vivo [36,73]. For example, we have shown that mechanical perturbation remodels the interface of an exceedingly stable complex (Kd = 10−14 M, koff = 1.8 × 10−6 s−1) formed between the bacterial antibiotic nuclease colicin E9 and its inhibitor, immunity protein 9 (Im9) so that dissociation occurs at a surprisingly low force (<20 pN) [73]. Examination of the N-terminal sequence of E9 (through which force or remodelling is applied or carried out in vivo) showed that this region docks against the remainder of the globular domain with little side-chain inter-digitation (see Figure 4). As described for protein L, this is ideal for transmitting mechanical signals to the binding interface at low force. Remodelling increases the off-rate a million-fold relative to that expected for a slip bond, allowing Im9 release and E9 activation at a biologically relevant rate upon binding to a competing organism.

Bottom Line: The effects of force on the biophysical properties of biological systems can be large and varied.As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results.Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

No MeSH data available.


Related in: MedlinePlus