Unfolding Force Research Articles

Mechanical unfolding of single filamin A (ABP-280) molecules detected by atomic force microscopy

Filamin A (ABP-280), which is an actin-binding protein of 560 kDa as a dimer, can, together with actin filaments, produce an isotropic cross-linked three-dimensional network (actin/filamin A gel) that plays an important role in mechanical responses of cells in processes such as maintenance of membrane stability and translational locomotion. In this study, we investigated the mechanical properties of single filamin A molecules using atomic force microscopy. In force–extension curves, we observed sawtooth patterns corresponding to the unfolding of individual immunoglobulin (Ig)-fold domains of filamin A. At a pulling speed of 0.37 μm/s, the unfolding interval was sharply distributed around 30 nm, while the unfolding force ranged from 50 to 220 pN. This wide distribution of the unfolding force can be explained by variation in values of activation energy and the width of activation barrier of 24 Ig-fold domains of the filamin A at the unfolding transition. This unfolding can endow filamin A with great extensibility. The refolding of the unfolded chain of filamin A occurred when the force applied to the protein was reduced to near zero, indicating that its unfolding is reversible. Based on these results, we discuss here the physiological implications of the mechanical properties of single filamin A molecules.

Modeling AFM-Induced PEVK Extension and the Reversible Unfolding of Ig/FNIII Domains in Single and Multiple Titin Molecules

Molecular elasticity is associated with a select number of polypeptides and proteins, such as titin, Lustrin A, silk fibroin, and spider silk dragline protein. In the case of titin, the globular (Ig) and non-globular (PEVK) regions act as extensible springs under stretch; however, their unfolding behavior and force extension characteristics are different. Using our time-dependent macroscopic method for simulating AFM-induced titin Ig domain unfolding and refolding, we simulate the extension and relaxation of hypothetical titin chains containing Ig domains and a PEVK region. Two different models are explored: 1) a series-linked WLC expression that treats the PEVK region as a distinct entropic spring, and 2) a summation of N single WLC expressions that simulates the extension and release of a discrete number of parallel titin chains containing constant or variable amounts of PEVK. In addition to these simulations, we also modeled the extension of a hypothetical PEVK domain using a linear Hooke's spring model to account for “enthalpic” contributions to PEVK elasticity. We find that the modified WLC simulations feature chain length compensation, Ig domain unfolding/refolding, and force-extension behavior that more closely approximate AFM, laser tweezer, and immunolocalization experimental data. In addition, our simulations reveal the following: 1) PEVK extension overlaps with the onset of Ig domain unfolding, and 2) variations in PEVK content within a titin chain ensemble lead to elastic diversity within that ensemble.

Native topology determines force-induced unfolding pathways in globular proteins.

Single-molecule manipulation techniques reveal that stretching unravels individually folded domains in the muscle protein titin and the extracellular matrix protein tenascin. These elastic proteins contain tandem repeats of folded domains with beta-sandwich architecture. Herein, we propose by stretching two model sequences (S1 and S2) with four-stranded beta-barrel topology that unfolding forces and pathways in folded domains can be predicted by using only the structure of the native state. Thermal refolding of S1 and S2 in the absence of force proceeds in an all-or-none fashion. In contrast, phase diagrams in the force-temperature (f,T) plane and steered Langevin dynamics studies of these sequences, which differ in the native registry of the strands, show that S1 unfolds in an allor-none fashion, whereas unfolding of S2 occurs via an obligatory intermediate. Force-induced unfolding is determined by the native topology. After proving that the simulation results for S1 and S2 can be calculated by using native topology alone, we predict the order of unfolding events in Ig domain (Ig27) and two fibronectin III type domains ((9)FnIII and (10)FnIII). The calculated unfolding pathways for these proteins, the location of the transition states, and the pulling speed dependence of the unfolding forces reflect the differences in the way the strands are arranged in the native states. We also predict the mechanisms of force-induced unfolding of the coiled-coil spectrin (a three-helix bundle protein) for all 20 structures deposited in the Protein Data Bank. Our approach suggests a natural way to measure the phase diagram in the (f,C) plane, where C is the concentration of denaturants.

Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme.

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 +/- 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

Unfolding forces of titin and fibronectin domains directly measured by AFM.

AFM-based Single Molecule Force Spectroscopy provides a new tool for probing the mechanical properties of single molecules. In this chapter we show that the unfolding forces of single protein domains can be directly measured. Unfolding forces give new insight into protein stability that cannot be deduced from thermodynamic measurements. A comparison between the unfolding forces measured in Ig domains of the muscle protein titin and those measured in fibronectin Type III domains reveals an extraordinarily high stability of titin domains.

Structural Forces in Biomolecules

Abstract The high spatial resolution and the force sensitivity of the Atomic Force Microscope have made it possible to perform mechanical experiments with single molecules. These experiments give direct access to the forces that stabilize biomolecule structure. In a variety of examples we show how different molecular interactions determine the mechanical stability of polysaccharides, proteins, DNA and other biomolecules. Under the influence of a stretching force of around 750 pN the polysaccharide dextran undergoes a transition into a stretched conformation during which bond angles flip into a new conformation. Molecular dynamics simulations corroborated this conformational change. Proteins fold into compact domains. Especially for structural proteins the mechanical stability of these domains is of importance. We show that the immunoglobulin and fibronectin III domains of the muscle protein titin exhibit exceptionally high unfolding forces (150-250 pN) when stretched at speeds of normal muscle operation (Fig. 1).

Steered molecular dynamics simulation of conformational changes of immunoglobulin domain I27 interprete atomic force microscopy observations

Atomic force microscopy and steered molecular dynamics investigations of the response of so-called mechanical proteins like titin, tenascin or their individual immunoglobulin and fibronectin type III domains have lead to qualitative insights about the relationship between the β sandwich domain architecture and the function of this class of proteins. The proteins, linear segments of up to hundreds of domains, through strain induced shape changes, unfolding and refolding, maintain order and elasticity in cellular systems over a nearly tenfold length scale. In this paper we develop a steered molecular dynamics description of the response of the titin immunoglobulin domain I27 at the onset of domain unfolding in quantitative agreement with AFM observations. We show that if forces stronger than 50 pN are applied to the terminal ends the two hydrogen bonds between the antiparallel A and B β strands break with a concomitant 6–7 Å elongation of the protein. If forces strong enough to unfold the domain are applied, the protein is halted in this initial extension until the set of all six hydrogen bonds connecting strands A′ and G break simultaneously. This behavior is accounted for by a barrier separating folded and unfolded states, the shape of which is consistent with AFM and chemical denaturation data. We also demonstrate that steered molecular dynamics simulations which induce unfolding through slow pulling (speed 0.1 Å/ps) predict unfolding forces that are within a factor of two within force values extrapolated from AFM observations.

Forced unfolding of fibronectin type 3 modules: an analysis by biased molecular dynamics simulations

Titin, an important constituent of vertebrate muscles, is a protein of the order of a micrometer in length in the folded state. Atomic force microscopy and laser tweezer experiments have been used to stretch titin molecules to more than ten times their folded lengths. To explain the observed relation between force and extension, it has been suggested that the immunoglobulin and fibronectin domains unfold one at a time in an all-or-none fashion. We use molecular dynamics simulations to study the forced unfolding of two different fibronectin type 3 domains (the ninth, 9Fn3, and the tenth, 10Fn3, from human fibronectin) and of their heterodimer of known structure. An external biasing potential on the N to C distance is employed and the protein is treated in the polar hydrogen representation with an implicit solvation model. The latter provides an adiabatic solvent response, which is important for the nanosecond unfolding simulation method used here. A series of simulations is performed for each system to obtain meaningful results. The two different fibronectin domains are shown to unfold in the same way along two possible pathways. These involve the partial separation of the “β-sandwich”, an essential structural element, and the unfolding of the individual sheets in a stepwise fashion. The biasing potential results are confirmed by constant force unfolding simulations. For the two connected domains, there is complete unfolding of one domain ( 9Fn3) before major unfolding of the second domain ( 10Fn3). Comparison of different models for the potential energy function demonstrates that the dominant cohesive element in both proteins is due to the attractive van der Waals interactions; electrostatic interactions play a structural role but appear to make only a small contribution to the stabilization of the domains, in agreement with other studies of β-sheet stability. The unfolding forces found in the simulations are of the order of those observed experimentally, even though the speed of the former is more than six orders of magnitude greater than that used in the latter.

Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles

Spectrin repeats fold into triple helical coiled-coils comprising ∼106 amino acid residues. Using an AFM-related technique we measured the force required to mechanically unfold these repeats to be 25 to 35 pN. Under tension, individual spectrin repeats unfold independently and in an all-or-none process. The dependence of the unfolding forces on the pulling speed reveals that the corresponding unfolding potential is shallow with an estimated width of 1.5 nm. When the unfolded polypeptide strand is relaxed, several domains refold within less than a second. The unfolding forces of the α-helical spectrin domains are five to ten times lower than those found in domains with β-fold, like immunoglobulin or fibronectin Ill domains, where the tertiary structure is stabilized by hydrogen bonds between adjacent strands. This shows that the forces stabilizing the coiled-coil lead to a mechanically much weaker structure than multiple hydrogen-bonded β-sheets.

AFM, a tool for single-molecule experiments

Force versus elongation curves measured with AFM-related techniques reveal a detailed insight into material properties at the molecular level. Here an overview on different implementations of this technique is given. The lateral distribution of a receptor on a sample surface was determined by mapping the adhesion force with a corresponding ligand immobilized at the tip. A dextran pattern on a gold surface was measured by y mapping the rupture length of the polymer. Stretching single dextran molecules revealed a structural transition in the polysaccharide backbone at forces of 750 pN. The unfolding of individual domains in the modular protein titin was observed and information about the rate dependence of the unfolding forces was obtained.

The Mechanical Stability of Immunoglobulin and Fibronectin III Domains in the Muscle Protein Titin Measured by Atomic Force Microscopy

The domains of the giant muscle protein titin (connectin) provide interaction sites for other sarcomeric proteins and fulfill mechanical functions. In this paper we compare the unfolding forces of defined regions of different titin isoforms by single-molecule force spectroscopy. Constructs comprising six to eight immunoglobulin (Ig) domains located in the mechanically active I-band part of titin are compared to those containing fibronectin III (Fn3) and Ig domains from the A-band part. The high spatial resolution of the atomic force microscope allows us to detect differences in length as low as a few amino acids. Thus constructs of different lengths may be used as molecular rulers for structural comparisons with other modular proteins. The unfolding forces range between 150 and 300 pN and differ systematically between the constructs. Fn3 domains in titin exhibit 20% lower unfolding forces than Ig domains. Fn3 domains from tenascin, however, unfold at forces only half those of titin Fn3 domains. This indicates that the tightly folded titin domains are designed to maintain their structural integrity, even under the influence of stretching forces. Hence, at physiological forces, unfolding is unlikely unless the forces are applied for a long time (longer than minutes).

Complete Unfolding of the Titin Molecule under External Force

Titin (also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several earlier studies have implicated titin as playing a fundamental role in maintaining sarcomeric structural integrity and generating the passive force of muscle. The elastic properties of titin were characterized in recent single-molecule mechanical works that described the molecule as an entropic spring in which partial unfolding may take place at high forces during stretch and refolding at low forces during release. In the present work titin molecules were stretched using a laser tweezer with forces above 400 pN. The high external forces resulted in complete mechanical unfolding of the molecule, characterized by the disappearance of force hysteresis at high forces. Titin refolded following complete denaturation, as the hysteresis at low forces reappeared in subsequent stretch–release cycles. The broad force range throughout which unfolding occurred indicates that the various globular domains in titin require different unfolding forces due to differences in the activation energies for their unfolding.