The Molecular Mechanism Underlying Mechanical Anisotropy of the Protein GB1

Mechanical responses of elastic proteins are crucial for their biological function and nanotechnological use. Loading direction has been identified as one key determinant for the mechanical responses of proteins. However, it is not clear how a change in pulling direction changes the mechanical unfolding mechanism of the protein. Here, we combine protein engineering, single-molecule force spectroscopy, and steered molecular dynamics simulations to systematically investigate the mechanical response of a small globular protein GB1. Force versus extension profiles from both experiments and simulations reveal marked mechanical anisotropy of GB1. Using native contact analysis, we relate the mechanically robust shearing geometry with concurrent rupture of native contacts. This clearly contrasts the sequential rupture observed in simulations for the mechanically labile peeling geometry. Moreover, we identify multiple distinct mechanical unfolding pathways in two loading directions. Implications of such diverse unfolding mechanisms are discussed. Our results may also provide some insights for designing elastomeric proteins with tailored mechanical properties.     

Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering

Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).     

The mechanical hierarchies of fibronectin observed with single-molecule AFM

Mechanically induced conformational changes in proteins such as fibronectin are thought to regulate the assembly of the extracellular matrix and underlie its elasticity and extensibility. Fibronectin contains a region of tandem repeats of up to 15 type III domains that play critical roles in cell binding and self-assembly. Here, we use single-molecule force spectroscopy to examine the mechanical properties of fibronectin (FN) and its individual FNIII domains. We found that fibronectin is highly extensible due to the unfolding of its FNIII domains. We found that the native FNIII region displays strong mechanical unfolding hierarchies requiring 80 pN of force to unfold the weakest domain and 200 pN for the most stable domain. In an effort to determine the identity of the weakest/strongest domain, we engineered polyproteins composed of an individual domain and measured their mechanical stability by single-protein atomic force microscopy (AFM) techniques. In contrast to chemical and thermal measurements of stability, we found that the tenth FNIII domain is mechanically the weakest and that the first and second FNIII domains are the strongest. Moreover, we found that the first FNIII domain can acquire multiple, partially folded conformations, and that their incidence is modulated strongly by its neighbor FNIII domain. The mechanical hierarchies of fibronectin demonstrated here may be important for the activation of fibrillogenesis and matrix assembly.     

Mechanical Characterization of Protein L in the Low-Force Regime by Electromagnetic Tweezers/Evanescent Nanometry

Mechanical manipulation at the single molecule level of proteins exhibiting mechanical stability poses a technical challenge that has been almost exclusively approached by atomic force microscopy (AFM) techniques. However, due to mechanical drift limitations, AFM techniques are restricted to experimental recordings that last less than a minute in the high-force regime. Here we demonstrate a novel combination of electromagnetic tweezers and evanescent nanometry that readily captures the forced unfolding trajectories of protein L at pulling forces as low as 10 ∼ 15 pN. Using this approach, we monitor unfolding and refolding cycles of the same polyprotein for a period of time longer than 30 min. From such long-lasting recordings, we obtain ensemble averages of unfolding step sizes and rates that are consistent with single-molecule AFM data obtained at higher stretching forces. The unfolding kinetics of protein L at low stretching forces confirms and extends the observations that the mechanical unfolding rate is exponentially dependent on the pulling force within a wide range of stretching forces spanning from 13 pN up to 120 pN. Our experiments demonstrate a novel approach for the mechanical manipulation of single proteins for extended periods of time in the low-force regime.     

The study of protein mechanics with the atomic force microscope.

The unfolding and folding of single protein molecules can be studied with an atomic force microscope (AFM). Many proteins with mechanical functions contain multiple, individually folded domains with similar structures. Protein engineering techniques have enabled the construction and expression of recombinant proteins that contain multiple copies of identical domains. Thus, the AFM in combination with protein engineering has enabled the kinetic analysis of the force-induced unfolding and refolding of individual domains as well as the study of the determinants of mechanical stability.     

Frequency modulation atomic force microscopy reveals individual intermediates associated with each unfolded I27 titin domain

In this study, we apply a dynamic atomic force microscopy (AFM) technique, frequency modulation (FM) detection, to the mechanical unfolding of single titin I27 domains and make comparisons with measurements made using the AFM contact or static mode method. Static mode measurements revealed the well-known force transition occurring at 100-120 pN in the first unfolding peak, which was less clear, or more often absent, in the subsequent unfolding peaks. In contrast, some FM-AFM curves clearly resolved a force transition associated with each of the unfolding peaks irrespective of the number of observed unfolded domains. As expected for FM-AFM, the frequency shift response of the main unfolding peaks and their intermediates could only be detected when the oscillation amplitudes used were smaller than the interaction lengths being measured. It was also shown that the forces measured for the dynamical interaction of the FM-AFM technique were significantly lower than those measured using the static mode. This study highlights the potential for using dynamic AFM for investigating biological interactions, including protein unfolding and the detection of novel unfolding intermediates.     

Unfolding mechanics of multiple OspA substructures investigated with single molecule force spectroscopy

We investigated mechanical unfolding of Borrelia burgdorferi outer surface protein A (OspA), a Lyme disease antigen containing a unique single-layer beta-sheet, with atomic force microscopy (AFM). We mechanically stretched a monomeric unit, rather than a tandem repeat, by pulling it from its N and C-terminal residues without using intervening polymer as a spacer. We detected two peaks in the force-extension profile before the final rupture of a fully extended polypeptide, which we interpreted as unfolding of multiple substructures in OspA. The double-peaked unfolding curves are consistent with results of previous thermodynamic studies showing two cooperative units in OspA. The mechanical unfolding processes were reversible, and the two substructures refolded within one second. Mutations near the boundary of the two thermodynamic cooperative units reduced the height of the first unfolding peak to undetectable levels and marginally affected the second one, indicating that the boundary between the two mechanical substructures is related to that previously assigned between the thermodynamic cooperative units. Based on a "worm-like chain" analysis of our AFM data, we propose a model for mechanical unfolding of OspA, where nearly a half of the chain is stretched with minimal resistive force, followed by sequential breakdown of C-terminal and N-terminal substructures. Based on these results, we discuss similarities and differences between mechanical and thermodynamic unfolding reactions of OspA. This work demonstrates that AFM study of monomeric proteins can elucidate details of the intramolecular mechanics of protein substructures.     

Single molecule force spectroscopy reveals a weakly populated microstate of the FnIII domains of tenascin

The native states of proteins exist as an ensemble of conformationally similar microstates. The fluctuations among different microstates are of great importance for the functions and structural stability of proteins. Here, we demonstrate that single molecule atomic force microscopy (AFM) can be used to directly probe the existence of multiple folded microstates. We used the AFM to repeatedly stretch and relax a recombinant tenascin fragment TNfnALL to allow the fibronectin type III (FnIII) domains to undergo repeated unfolding/refolding cycles. In addition to the native state, we discovered that some FnIII domains can refold from the unfolded state into a previously unrecognized microstate, N* state. This novel state is conformationally similar to the native state, but mechanically less stable. The native state unfolds at approximately 120 pN, while the N* state unfolds at approximately 50 pN. These two distinct populations of microstates constitute the ensemble of the folded states for some FnIII domains. An unfolded FnIII domain can fold into either one of the two microstates via two distinct folding routes. These results reveal the dynamic and heterogeneous picture of the folded ensemble for some FnIII domains of tenascin, which may carry important implications for the mechanical functions of tenascins in vivo.     

Unfolding a linker between helical repeats

In many multi-repeat proteins, linkers between repeats have little secondary structure and place few constraints on folding or unfolding. However, the large family of spectrin-like proteins, including alpha-actinin, spectrin, and dystrophin, share three-helix bundle, spectrin repeats that appear in crystal structures to be linked by long helices. All of these proteins are regularly subjected to mechanical stress. Recent single molecule atomic force microscopy (AFM) experiments demonstrate not only forced unfolding but also simultaneous unfolding of tandem repeats at finite frequency, which suggests that the contiguous helix between spectrin repeats can propagate a cooperative helix-to-coil transition. Here, we address what happens atomistically to the linker under stress by steered molecular dynamics simulations of tandem spectrin repeats in explicit water. The results for alpha-actinin repeats reveal rate-dependent pathways, with one pathway showing that the linker between repeats unfolds, which may explain the single-repeat unfolding pathway observed in AFM experiments. A second pathway preserves the structural integrity of the linker, which explains the tandem-repeat unfolding event. Unfolding of the linker begins with a splay distortion of proximal loops away from hydrophobic contacts with the linker. This is followed by linker destabilization and unwinding with increased hydration of the backbone. The end result is an unfolded helix that mechanically decouples tandem repeats. Molecularly detailed insights obtained here aid in understanding the mechanical coupling of domain stability in spectrin family proteins.     

Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation

Atomic force microscopy (AFM) experiments have provided intriguing insights into the mechanical unfolding of proteins such as titin I27 from muscle, but will the same be possible for proteins that are not physiologically required to resist force? We report the results of AFM experiments on the forced unfolding of barnase in a chimeric construct with I27. Both modules are independently folded and stable in this construct and have the same thermodynamic and kinetic properties as the isolated proteins. I27 can be identified in the AFM traces based on its previous characterization, and distinct, irregular low-force peaks are observed for barnase. Molecular dynamics simulations of barnase unfolding also show that it unfolds at lower forces than proteins with mechanical function. The unfolding pathway involves the unraveling of the protein from the termini, with much more native-like secondary and tertiary structure being retained in the transition state than is observed in simulations of thermal unfolding or experimentally, using chemical denaturant. Our results suggest that proteins that are not selected for tensile strength may not resist force in the same way as those that are, and that proteins with similar unfolding rates in solution need not have comparable unfolding properties under force.     

Protein-protein interaction regulates proteins' mechanical stability

Elastomeric proteins are molecular springs found not only in a variety of biological machines and tissues, but also in biomaterials of superb mechanical properties. Regulating the mechanical stability of elastomeric proteins is not only important for a range of biological processes, but also critical for the use of engineered elastomeric proteins as building blocks to construct nanomechanical devices and novel materials of well-defined mechanical properties. Here we demonstrate that protein-protein interactions can potentially serve as an effective means to regulate the mechanical properties of elastomeric proteins. We show that the binding of fragments of IgG antibody to a small protein, GB1, can significantly enhance the mechanical stability of GB1. The regulation of the mechanical stability of GB1 by IgG fragments is not through direct modification of the interactions in the mechanically key region of GB1; instead, it is accomplished via the long-range coupling between the IgG binding site and the mechanically key region of GB1. Although Fc and Fab bind GB1 at different regions of GB1, their binding to GB1 can increase the mechanical stability of GB1 significantly. Using alanine point mutants of GB1, we show that the amplitude of mechanical stability enhancement of GB1 by Fc does not correlate with the binding affinity, suggesting that binding affinity only affects the population of GB1/human Fc (hFc) complex at a given concentration of hFc, but does not affect the intrinsic mechanical stability of the GB1/hFc complex. Furthermore, our results indicate that the mechanical stability enhancement by IgG fragments is robust and can tolerate sequence/structural perturbation to GB1. Our results demonstrate that the protein-protein interaction is an efficient approach to regulate the mechanical stability of GB1-like proteins and we anticipate that this new methodology will help to develop novel elastomeric proteins with tunable mechanical stability and compliance.     

Steered molecular dynamics studies of titin I1 domain unfolding

The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging beta-strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B beta-strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A'- and G-strands. The disulfide bond also limits the maximum extension of I1 to approximately 220 A. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.     

Mechanical stability and differentially conserved physical–chemical properties of titin Ig‐domains

The mechanisms that determine mechanical stabilities of protein folds remain elusive. Our understanding of these mechanisms is vital to both bioengineering efforts and to the better understanding and eventual treatment of pathogenic mutations affecting mechanically important proteins such as titin. We present a new approach to analyze data from single‐molecule force spectroscopy for different domains of the giant muscle protein titin. The region of titin found in the I‐band of a sarcomere is composed of about 40 Ig‐domains and is exposed to force under normal physiological conditions and connects the free‐hanging ends of the myosin filaments to the Z‐disc. Recent single‐molecule force spectroscopy data show a mechanical hierarchy in the I‐band domains. Domains near the C‐terminus in this region unfold at forces two to three times greater than domains near the beginning of the I‐band. Though all of these Ig‐domains are thought to share a fold and topology common to members of the Ig‐like fold family, the sequences of neighboring domains vary greatly with an average sequence identity of only 25%. We examine in this study the relation of these unique mechanical stabilities of each I‐band Ig domain to specific, conserved physical–chemical properties of amino acid sequences in related Ig domains. We find that the sequences of each individual titin Ig domain are very highly conserved, with an average sequence identity of 79% across species that are divergent as humans, chickens, and zebra fish. This indicates that the mechanical properties of each domain are well conserved and tailored to its unique position in the titin molecule. We used the PCPMer software to determine the conservation of amino acid properties in titin Ig domains grouped by unfolding forces into “strong” and “weak” families. We found two motifs unique to each family that may have some role in determining the mechanical properties of these Ig domains. A detailed statistical analysis of properties of individual residues revealed several positions that displayed differentially conserved properties in strong and weak families. In contrast to previous studies, we find evidence that suggests that the mechanical stability of Ig domains is determined by several residues scattered across the β‐sandwich fold, and force sensitive residues are not only confined to the A′‐G region. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Ligand-modulated parallel mechanical unfolding pathways of maltose-binding proteins

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.     

Prediction of the translocation kinetics of a protein from its mechanical properties

Proteins are actively unfolded to pass through narrow channels in macromolecular complexes that catalyze protein translocation and degradation. Catalyzed unfolding shares many features that characterize the mechanical unfolding of proteins using the atomic force microscope (AFM). However, simulations of unfolding induced by the AFM and when a protein is translocated through a pore suggest that each process occurs by distinct pathways. The link, if any, between each type of unfolding, therefore, is not known. We show that the mechanical unfolding energy landscape of a protein, obtained using an atomistic molecular model, can be used to predict both the relative mechanical strength of proteins when unfolded using the AFM and when unfolded by translocation into a pore. We thus link the two processes and show that the import rate through a pore not only depends on the location of the initiation tag but also on the mechanical properties of the protein when averaged over all the possible geometries that are relevant for a given translocation initiation site.     

Mechanically unfolding protein L using a laser-feedback-controlled cantilever

Force spectroscopy using the atomic force microscope (AFM) can yield important information on the strength and lifetimes of the folded states of single proteins and their complexes when they are loaded with force. For example, by mechanically unfolding concatenated proteins at different velocities, a dynamic force spectrum can be built up that allows reconstruction of the energy landscape that the protein traverses during unfolding. To characterize fully the unfolding landscape, however, it is necessary both to explore the entire force spectrum and to characterize each species populated during unfolding. In the conventional AFM apparatus, force is applied to the protein construct through a compliant cantilever. This limits the dynamic range of the force spectrum that can be probed, and the cantilever recoil after unfolding may mask the presence of metastable intermediates. Here, we describe to our knowledge a new technique—constant-deflection AFM—in which the compliance of the AFM cantilever is removed. Using this technique, we show that protein L exhibits a more complex unfolding energy landscape than previously detected using the conventional technique. This technique is also able to detect the presence of a refolding intermediate whose formation is otherwise prevented by cantilever recoil.     

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).     

Single-molecule detection of phosphorylation-induced plasticity changes during ezrin activation

Ezrin-radixin-moesin protein family provides a regulated link between the cortical actin cytoskeleton and the plasma membrane. Phosphorylation of ezrin has been functionally linked to membrane dynamics and plasticity. Our recent study demonstrated that phosphorylation of the conserved T567 residue of ezrin alters the physiology of gastric parietal cells. However, the molecular mechanism of phosphorylation-induced ezrin activation has remained elusive. Here we use atomic force microscopy (AFM) to probe phosphorylation-mediated activation of ezrin in single molecules. The phospho-mimicking and non-phosphorylatable mutant ezrin proteins were generated and purified to homogeneity. Comparative analyses of two ezrin mutants by AFM demonstrate the unfolding of the N- and C-terminal domains upon the phospho-activation. To measure the physical force underlying the inter-domain contact during mechanical unfolding, we probed the defined region of ezrin using the N-terminal ezrin coated onto the AFM tip. Comparative force measurements indicate that T567 phosphorylation-induced unfolding of ezrin favors the inter-molecular association. Taken together, these results provide molecular illustration of phosphorylation elicited functional activation of ERM proteins and indicate that stimulus-induced protein conformational change can be used as a signaling mechanism orchestrating cellular dynamics.     

Single molecule force spectroscopy reveals that electrostatic interactions affect the mechanical stability of proteins

It is well known that electrostatic interactions play important roles in determining the thermodynamic stability of proteins. However, the investigation into the role of electrostatic interactions in mechanical unfolding of proteins has just begun. Here we used single molecule atomic force microscopy techniques to directly evaluate the effect of electrostatic interactions on the mechanical stability of a small protein GB1. We engineered a bi-histidine motif into the force-bearing region of GB1. By varying the pH, histidine residues can switch between protonated and deprotonated states, leading to the change of the electrostatic interactions between the two histidine residues. We found that the mechanical unfolding force of the engineered protein decreased by ∼34% (from 115 pN to 76 pN) on changing the pH from 8.5 to 3, due to the increased electrostatic repulsion between the two positively charged histidines at acidic pH. Our results demonstrated that electrostatic interactions can significantly affect the mechanical stability of elastomeric proteins, and modulating the electrostatic interactions of key charged residues can become a promising method for regulating the mechanical stability of elastomeric proteins.     

Viscoelastic study of the mechanical unfolding of a protein by AFM

We have applied a dynamic force modulation technique to the mechanical unfolding of a homopolymer of immunoglobulin (Ig) domains from titin, (C47S C63S I27)5, [(I27)5] to determine the viscoelastic response of single protein molecules as a function of extension. Both the stiffness and the friction of the homopolymer system show a sudden decrease when a protein domain unfolds. The decrease in measured friction suggests that the system is dominated by the internal friction of the (I27)5 molecule and not solvent friction. In the stiffness-extension spectrum we detected an abrupt feature before each unfolding event, the amplitude of which decreased with each consecutive unfolding event. We propose that these features are a clear indication of the formation of the known unfolding intermediate of I27, which has been observed previously in constant velocity unfolding experiments. This simple force modulation AFM technique promises to be a very useful addition to constant velocity experiments providing detailed viscoelastic characterization of single molecules under extension.