Dynamic force spectroscopy of the digoxigenin–antibody complex

Neuert, Gregor; Albrecht, Christian; Pamir, E.; Gaub, Hermann E.

doi:10.1016/j.febslet.2005.12.052

Cited by 141 publications

(156 citation statements)

References 38 publications

Supporting

Mentioning

140

Contrasting

Unclassified

Order By: Relevance

“…Supporting this interpretation, the measured transitionstate distance d of Ϸ0.6 nm that the center of mass of the double-headed molecule has to be moved for the limiting transition to occur (see Fig. 3D) is comparable to the distances measured for breaking molecular interfaces in proteins or antibody-antigen interactions (30,31).…”

Section: Discussionsupporting

confidence: 53%

Myosin-V is a mechanical ratchet

Gebhardt

Clemen

Jaud

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

135

192

View full text Add to dashboard Cite

Myosin-V is a linear molecular motor that hydrolyzes ATP to move processively toward the plus end of actin filaments. Motion of this motor under low forces has been studied recently in various single-molecule assays. In this paper we show that myosin-V reacts to high forces as a mechanical ratchet. High backward loads can induce rapid and processive backward steps along the actin filament. This motion is completely independent of ATP binding and hydrolysis. In contrast, forward forces cannot induce ATP-independent forward steps. We can explain this pronounced mechanical asymmetry by a model in which the strength of actin binding of a motor head is modulated by the lever arm conformation. Knowledge of the complete force-velocity dependence of molecular motors is important to understand their function in the cellular environment.backward movement ͉ molecular motor ͉ optical tweezers ͉ asymmetry ͉ kinesin C lass-V myosins are two-headed linear molecular motors involved in various intracellular transport processes that move processively and directionally toward the plus end of actin filaments (1-4). The energy required for forward motion is supplied by hydrolysis of ATP (1, 5). During each forward step both heads of a myosin-V motor undergo a coordinated chemomechanical cycle, which results in a hand-over-hand stepping mechanism (6, 7). After release of ADP in the trailing head, ATP can bind, and this head detaches from the filament. Now, the leading head can perform the power stroke of its lever arm (8). Subsequently, the now-forward head can rebind to the filament. The mechanism that prevents premature ADP release from the leading head in a two-head-bound myosin is believed to be based on intramolecular strain (9-11).Apart from myosin-V, forward motion has been studied extensively for many linear and rotary motors (12)(13)(14)(15)(16)(17)(18)(19). For most of these motors tight coupling of forward motion to ATP hydrolysis has been reported (6,20,21). In contrast, the modes of force-induced backward motion seem to be quite diverse in the different motor systems. Whereas for the F 1 -ATPase the hydrolysis cycle is completely reversible, and forced backward rotation can lead to ATP synthesis (22, 23), backward steps of the linear motor kinesin have been shown to be tightly coupled to ATP binding (24,25). In kinesin, backward forces lead to a decrease of the intrinsic forward bias of a step. In the present study, we investigate force-driven motion of myosin-V by using an optical trap with force feedback control in which we observe a forceinduced mode of backward motion distinct from kinesin and completely independent from the ATP cycle. ResultsIn a first set of experiments we tested motor velocities under various high loads in forward and backward direction at 1 M ATP. Forces in the direction of unloaded movement (forward) of myosin-V and forces opposing the unloaded movement (backward) of a motor attached to a trapped polystyrene bead were applied by moving a piezo-driven microscope stage parallel to a surface-anchor...

show abstract

Section: Discussionsupporting

confidence: 53%

Myosin-V is a mechanical ratchet

Gebhardt

Clemen

Jaud

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

135

192

View full text Add to dashboard Cite

show abstract

“…For a lower loading rate the rupture force was found to be lower. For a-DIG/DIG a rupture force of 30-40 pN was found for a loading rate of 10 3 pN/s (Neuert et al, 2006), which corresponds well to our data, and would suggest that we indeed observe single a-DIG/DIG binding events.…”

Section: Localization Of Dig Molecules On Asupporting

confidence: 86%

Combining optical tweezers and scanning probe microscopy to study DNA–protein interactions

Huisstede

Subramaniam

Bennink

2006

Microscopy Res & Technique

View full text Add to dashboard Cite

We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers, while a probe is scanned along the molecule. Interaction forces between the probe and the sample are measured with the optical tweezers. The instrument thus enables us to correlate mechanical and functional properties of bound proteins with the tension within the DNA molecule. The typical friction force between a micropipette used as probe and a naked DNA molecule was found to be <1 pN. A 16 micro m DNA molecule with approximately 10-15 digoxygenin (DIG) molecules located over a 90 nm range in the middle of the DNA was used as a model system. By scanning with an antidigoxygenin (alpha-DIG) antibody-coated pipette we were able to localize these sites by exploiting the high binding affinity between this antibody-antigen pair. The estimated experimental resolution assuming an infinitesimally thin and rigid probe and a single alpha-DIG/DIG bond was 15 nm.

show abstract

“…5 B and C), consistent with mechanochemistry taking place as the cantilever breaks a covalent bond when it retracts (18). The median breakage force for the covalent interaction was 1.9 nN, which is >20 times stronger than streptavidin-biotin or antibody-antigen interactions (20,21). C-N and C-C bonds are not predicted to break until 5 nN (18), so it is likely that was not the isopeptide bond, nor another bond in the protein which broke, but rather a weaker link in the chain, such as a C-S bond linking the proteins to the cantilever/ bead.…”

Section: Resultsmentioning

confidence: 55%

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Zakeri

Fierer

Çelik

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

1,264

1,581

View full text Add to dashboard Cite

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures. agging with peptides (e.g., HA, myc, FLAG, His 6 ) is one of the most common ways to detect, purify, or immobilize proteins (1-4). Peptides are very useful minimally disruptive probes (5) but they are also "slippery"-antibodies or other proteins typically bind peptides with low affinity and poor mechanical strength (6-9). We sought to form a rapid covalent bond to a peptide tag without the use of chemical modification, artificial amino acids, or cysteines (disulfide bond formation is reversible and restricted to particular cellular locations).It has recently been found that Streptococcus pyogenes, like many other Gram-positive bacteria, contains extracellular proteins stabilized by spontaneous intramolecular isopeptide bonds (10). Here we explored the second immunoglobulin-like collagen adhesin domain (CnaB2) from the fibronectin binding protein FbaB, found in invasive strains of S. pyogenes (11,12) and essential for phagocytosis-like uptake of the bacteria by endothelial cells (13). CnaB2 contains a single isopeptide bond conferring exceptional stability: CnaB2 remains folded even at pH 2 or up to 100°C (12). By splitting CnaB2 into peptide and protein fragments, followed by rational modification of the parts, we developed a peptide tag of 13 amino acids that rapidly formed a covalent bond with its protein partner (138 amino acids, 15 kDa) and characterized the conditions for reaction, cellular specificity of bond formation, and resilience of the reacted product.

show abstract

Dynamic force spectroscopy of the digoxigenin–antibody complex

Cited by 141 publications

References 38 publications

Myosin-V is a mechanical ratchet

Myosin-V is a mechanical ratchet

Combining optical tweezers and scanning probe microscopy to study DNA–protein interactions

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Contact Info

Product

Resources

About