Enhancing the mechanical stability of proteins through a cocktail approach

Rationally enhancing the mechanical stability of proteins remains a challenge in the field of single molecule force spectroscopy. Here we demonstrate that it is feasible to use a “cocktail” approach for combining more than one approach to enhance significantly the mechanical stability of proteins in an additive fashion. As a proof of principle, we show that metal chelation and protein-protein interaction can be combined to enhance the unfolding force of a protein to ∼450 pN, which is >3 times of its original value. This is also higher than the mechanical stability of most of proteins studied so far. We also extend such a cocktail concept to combine two different metal chelation sites to enhance protein mechanical stability. This approach opens new avenues to efficiently regulating the mechanical properties of proteins, and should be applicable to a wide range of elastomeric proteins.     

The effects of amino acid replacements of glycine 20 on conformational stability and catalysis of staphylococcal nuclease

Staphylococcal nuclease (SNase) is a well-established model for protein folding studies. Its three-dimensional structure has been determined. The enzyme, Ca2+, and DNA or RNA substrate form a ternary complex. Glycine 20 is the second position of the first beta-turn of SNase, which may serve as the folding initiation site for the SNase polypeptide. To study the role of Gly20 in the conformational stability and catalysis of SNase, three mutants, in which Gly20 was replaced by alanine, valine, or isoleucine, were constructed and studied by using circular dichroism spectra, intrinsic and ANS-binding fluorescence spectra, stability and activity assays. The mutations have little effect on the conformational integrity of the mutants. However, the catalytic activity is reduced drastically by the mutations, and the stability of the protein is progressively decreased in the order G20A相似文献   

Cross-Species Mechanical Fingerprinting of Cardiac Myosin Binding Protein-C

Cardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin III (FNIII)-like domains along with a unique regulatory sequence referred to as the MyBP-C motif or M-domain. We previously used atomic force microscopy to investigate the mechanical properties of murine cMyBP-C expressed using a baculovirus/insect cell expression system. Here, we investigate whether the mechanical properties of cMyBP-C are conserved across species by using atomic force microscopy to manipulate recombinant human cMyBP-C and native cMyBP-C purified from bovine heart. Force versus extension data obtained in velocity-clamp experiments showed that the mechanical response of the human recombinant protein was remarkably similar to that of the bovine native cMyBP-C. Ig/Fn-like domain unfolding events occurred in a hierarchical fashion across a threefold range of forces starting at relatively low forces of ∼50 pN and ending with the unfolding of the highest stability domains at ∼180 pN. Force-extension traces were also frequently marked by the appearance of anomalous force drops suggestive of additional mechanical complexity such as structural coupling among domains. Both recombinant and native cMyBP-C exhibited a prominent segment ∼100 nm-long that could be stretched by forces <50 pN before the unfolding of Ig- and FN-like domains. Combined with our previous observations of mouse cMyBP-C, these results establish that although the response of cMyBP-C to mechanical load displays a complex pattern, it is highly conserved across species.     

Ligand binding modulates the mechanical stability of dihydrofolate reductase

We use single-molecule force spectroscopy to demonstrate that the mechanical stability of the enzyme dihydrofolate reductase (DHFR) is modulated by ligand binding. In the absence of bound ligands, DHFR extends at very low forces, averaging 27 pN, without any characteristic mechanical fingerprint. By contrast, in the presence of micromolar concentrations of the ligands methotrexate, nicotinamide adenine dihydrogen phosphate, or dihydrofolate, much higher forces are required (82 +/- 18 pN, 98 +/- 15 pN, and 83 +/- 16 pN, respectively) and a characteristic fingerprint is observed in the force-extension curves. The increased mechanical stability triggered by these ligands is not additive. Our results explain the large reduction in the degradation rate of DHFR, in the presence of its ligands. Our observations support the view that the rate-limiting step in protein degradation by adenosine triphosphate-dependent proteases is the mechanical unfolding of the target protein.     

Cofilin increases the bending flexibility of actin filaments: implications for severing and cell mechanics

We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven, two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 μm, corresponding to a flexural rigidity of 0.040 pN μm². Cofilin binding lowers the persistence length ∼5-fold to a value of 2.2 μm and the filament flexural rigidity to 0.0091 pN μm². That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics, together with our previous analysis of torsional stiffness, provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding, and suggest that the overall mechanical and force-producing properties of cells can be modulated by cofilin activity.     

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.     

Differential mechanical stability of filamin A rod segments

Prompted by recent reports suggesting that interaction of filamin A (FLNa) with its binding partners is regulated by mechanical force, we examined mechanical properties of FLNa domains using magnetic tweezers. FLNa, an actin cross-linking protein, consists of two subunits that dimerize through a C-terminal self-association domain. Each subunit contains an N-terminal spectrin-related actin-binding domain followed by 24 immunoglobulinlike (Ig) repeats. The Ig repeats in the rod 1 segment (repeats 1–15) are arranged as a linear array, whereas rod 2 (repeats 16–23) is more compact due to interdomain interactions. In the rod 1 segment, repeats 9–15 augment F-actin binding to a much greater extent than do repeats 1–8. Here, we report that the three segments are unfolded at different forces under the same loading rate. Remarkably, we found that repeats 16–23 are susceptible to forces of ∼10 pN or even less, whereas the repeats in the rod 1 segment can withstand significantly higher forces. The differential force response of FLNa Ig domains has broad implications, since these domains not only support the tension of actin network but also interact with many transmembrane and signaling proteins, mostly in the rod 2 segment. In particular, our finding of unfolding of repeats 16–23 at ∼10 pN or less is consistent with the hypothesized force-sensing function of the rod 2 segment in FLNa.     

Mechanical Distortion of Single Actin Filaments Induced by External Force: Detection by Fluorescence Imaging

Actin is a major component of the cytoskeleton that transmits mechanical stress in both muscle and nonmuscle cells. As the first step toward developing a “bio-nano strain gauge” that would be able to report the mechanical stress imposed on an actin filament, we quantitatively examined the fluorescence intensity of dyes attached to single actin filaments under various tensile forces (5-20 pN). Tensile force was applied via two optically trapped plastic beads covalently coated with chemically modified heavy meromyosin molecules that were attached to both end regions of an actin filament. As a result, we found that the fluorescence intensity of an actin filament, where 20% of monomers were labeled with tetramethylrhodamine (TMR)-5-maleimide at Cys³⁷⁴ and the filamentous structure was stabilized with nonfluorescent phalloidin, decreased by ∼6% per 10 pN of the applied force, whereas the fluorescence intensity of an actin filament labeled with either BODIPY TMR cadaverin-iodoacetamide at Cys³⁷⁴ or rhodamine-phalloidin showed only an ∼2% decrease per 10 pN of the applied force. On the other hand, spectroscopic measurements of actin solutions showed that the fluorescence intensity of TMR-actin increased 1.65-fold upon polymerization (G-F transformation), whereas that of BODIPY-actin increased only 1.06-fold. These results indicate that the external force distorts the filament structure, such that the microenvironment around Cys³⁷⁴ approaches that in G-actin. We thus conclude that the fluorescent dye incorporated into an appropriate site of actin can report the mechanical distortion of the binding site, which is a necessary condition for the bio-nano strain gauge.     

The effect of temperature on mechanical resistance of the native and intermediate states of I27

We investigated the effect of temperature on the mechanical unfolding of I27 from human cardiac titin, employing a custom-built temperature control device for single-molecule atomic force microscopy measurement. A sawtooth pattern was observed in the force curves where each force peak reports on the unfolding of an I27 domain. In early unfolding events, we observed a “hump-like” deviation due to the detachment of β-strand A from each I27 domain. The force at which the humps appear was ∼130 pN and showed no temperature dependence, at least in the temperature range of 2°C-30°C. The hump structure was successfully analyzed with a two-state worm-like chain model, and the Gibbs free energy difference of the detachment reaction was estimated to be 11.6 ± 0.58 kcal/mol and found to be temperature independent. By contrast, upon lowering the temperature, the mean unfolding force from the partly unfolded intermediate state was found to markedly increase and the unfolding force distribution to broaden significantly, suggesting that the distance (x_u) between the folded and transition states in the energy landscape along the pulling direction is decreased. These results suggest that the local structure of β-strand A are stabilized by topologically simple local hydrogen-bond network and that the temperature does not affect the detachment reaction thermodynamically and kinetically, whereas the interaction between the β-strands A′ and G, which is a critical region for its mechanical stability, is strongly dependent on the temperature.     

The C2 Domains of Human Synaptotagmin 1 Have Distinct Mechanical Properties

Synaptotagmin 1 (Syt1) is the Ca⁺² receptor for fast, synchronous vesicle fusion in neurons. Because membrane fusion is an inherently mechanical, force-driven event, Syt1 must be able to adapt to the energetics of the fusion apparatus. Syt1 contains two C2 domains (C2A and C2B) that are homologous in sequence and three-dimensional in structure; yet, a number of observations have suggested that they have distinct biochemical and biological properties. In this study, we analyzed the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ∼100 pN was identified as C2B and the second peak of ∼50 pN as C2A. Furthermore, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of the C2A domain and modulate the affinity for soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), phospholipids, and Ca⁺².     

DNA-enzyme conjugate with a weak inhibitor that can specifically detect thrombin in a homogeneous medium

We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA–enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5′ end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni²⁺ complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by increasing the local concentration of the weak inhibitor by the hybridization of a 3′-end fluorescein-modified DNA. The catalytic activity of the DNA aptamer–enzyme conjugate showed a dose-dependent response to thrombin, indicating that the GST activity was clearly recovered by the binding of the DNA aptamer to thrombin. The current system enables the sensitive and specific detection of thrombin simply by measuring the enzymatic activity in a homogeneous medium.     

Molecular mechanics of filamin's rod domain

Rearrangement of the actin cytoskeleton is integral to cell shape and function. Actin-binding proteins, e.g., filamin, can naturally contribute to the mechanics and function of the actin cytoskeleton. The molecular mechanical bases for filamin's function in actin cytoskeletal reorganization are examined here using molecular dynamics simulations. Simulations are performed by applying forces ranging from 25 pN to 125 pN for 2.5 ns to the rod domain of filamin. Applying small loads (∼25 pN) to filamin's rod domain supplies sufficient energy to alter the conformation of the N-terminal regions of the rod. These forces break local hydrogen bond coordination often enough to allow side chains to find new coordination partners, in turn leading to drastic changes in the conformation of filamin, for example, increasing the hydrophobic character of the N-terminal rod region and, alternatively, activating the C-terminal region to become increasingly stiff. These changes in conformation can lead to changes in the affinity of filamin for its binding partners. Therefore, filamin can function to transduce mechanical signals as well as preserve topology of the actin cytoskeleton throughout the rod domain.     

HIV-1 integrase strand transfer inhibitors stabilize an integrase-single blunt-ended DNA complex

Integration of human immunodeficiency virus cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt ends to form the synaptic complex, which is the intermediate in the concerted integration pathway. The synaptic complex is inactivated by strand transfer inhibitors (STI) with IC₅₀ values of ∼ 20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on a native agarose gel that was produced in the presence of > 200 nM STI, termed the IN-single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus, producing an ∼ 32-bp DNase I protective footprint. In the presence of raltegravir (RAL), MK-2048, and L-841,411, IN incorporated ∼ 20-25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤ 5% of input DNA). The formation of the ISD complex was not dependent on 3′OH processing, and the DNA was predominantly blunt ended in the complex. The RAL-resistant IN mutant N155H weakly forms the ISD complex in the presence of RAL at ∼ 25% level of wild-type IN. In contrast, MK-2048 and L-841,411 produced ∼ 3-fold to 5-fold more ISD than RAL with N155H IN, which is susceptible to these two inhibitors. The results suggest that STI are slow-binding inhibitors and that the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex.     

Dissociation of Bimolecular αIIbβ3-Fibrinogen Complex under a Constant Tensile Force

The regulated ability of integrin αIIbβ3 to bind fibrinogen plays a crucial role in platelet aggregation, adhesion, and hemostasis. Employing an optical-trap-based electronic force clamp, we studied the thermodynamics and kinetics of αIIbβ3-fibrinogen bond formation and dissociation under constant unbinding forces, mimicking the forces of physiologic blood shear on a thrombus. The distribution of bond lifetimes was bimodal, indicating that the αIIbβ3-fibrinogen complex exists in two bound states with different mechanical stability. The αIIbβ3 antagonist, abciximab, inhibited binding without affecting the unbinding kinetics, whereas Mn²⁺ biased the αIIbβ3-fibrinogen complex to the strong bound state with reduced off-rate. The average bond lifetimes decreased exponentially with increasing pulling force from ∼5 pN to 50 pN, suggesting that in this force range the αIIbβ3-fibrinogen interactions are classical slip bonds. We found no evidence for catch bonds, which is consistent with the known lack of shear-enhanced platelet adhesion on fibrinogen-coated surfaces. Taken together, these data provide important quantitative and qualitative characteristics of αIIbβ3-fibrinogen binding and unbinding that underlie the dynamics of platelet adhesion and aggregation in blood flow.     

On the mechanical stabilization of filopodia

We studied force-induced elongation of filopodia by coupling magnetic tweezers to the tip through the bacterial coat protein invasin, which couples the force generator to the actin bundles (through myosin X), thus impeding the growth of the actin plus end. Single force pulses (15–30 s) with amplitudes between 20 and 600 pN and staircase-like force scenarios (amplitudes, ∼50 pN; step widths, 30 s) were applied. In both cases, the responses consist of a fast viscoelastic deflection followed by a linear flow regime. The deflections are reversible after switching off the forces, suggesting a mechanical memory. The elongation velocity exhibits an exponential distribution (half-width <v_1/2>, ∼0.02 μm s⁻¹) and did not increase systematically with the force amplitudes. We estimate the bending modulus (0.4 × 10⁻²³ J m) and the number of actin filaments (∼10) by analyzing filopodium bending fluctuations. Sequestering of intracellular Ca²⁺ by BAPTA caused a strong reduction in the amplitude of elongation, whereas latrunculin A resulted in loss of the elastic response. We attribute the force-independent velocity to the elongation of actin bundles enabled by the force-induced actin membrane uncoupling and the reversibility by the treadmilling mechanism and an elastic response.     

Probing the active site of the hepatitis C virus serine protease by fluorescence resonance energy transfer

A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.     

Mechanical unfolding of cardiac myosin binding protein-C by atomic force microscopy

Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.     

Stabilization provided by neighboring strands is critical for the mechanical stability of proteins

Single-molecule force spectroscopy studies and steered molecular dynamics simulations have revealed that protein topology and pulling geometry play important roles in determining the mechanical stability of proteins. Most studies have focused on local interactions that are associated with the force-bearing β-strands. Interactions mediated by neighboring strands are often overlooked. Here we use Top7 and barstar as model systems to illustrate the critical importance of the stabilization effect provided by neighboring β-strands on the mechanical stability. Using single-molecule atomic force microscopy, we showed that Top7 and barstar, which have similar topology in their force-bearing region, exhibit vastly different mechanical-stability characteristics. Top7 is mechanically stable and unfolds at ∼150 pN, whereas barstar is mechanically labile and unfolds largely below 50 pN. Steered molecular dynamics simulations revealed that stretching force peels one force-bearing strand away from barstar to trigger unfolding, whereas Top7 unfolds via a substructure-sliding mechanism. This previously overlooked stabilization effect from neighboring β-strands is likely to be a general mechanism in protein mechanics and can serve as a guideline for the de novo design of proteins with significant mechanical stability and novel protein topology.     

Integrin-Generated Forces Lead to Streptavidin-Biotin Unbinding in Cellular Adhesions

The interplay between chemical and mechanical signals plays an important role in cell biology, and integrin receptors are the primary molecules involved in sensing and transducing external mechanical cues. We used integrin-specific probes in molecular tension fluorescence microscopy to investigate the pN forces exerted by integrin receptors in living cells. The molecular tension fluorescence microscopy probe consisted of a cyclic Arg-Gly-Asp-D-Phe-Lys(Cys) (cRGDfK(C)) peptide tethered to the terminus of a polyethylene glycol polymer that was attached to a surface through streptavidin-biotin linkage. A fluorescence resonance energy transfer mechanism was used to visualize tension-driven extension of the polymer. Surprisingly, we found that integrin receptors dissociate streptavidin-biotin tethered ligands in focal adhesions within 60 min of cell seeding. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ∼10⁶ - 10⁸ times larger than that of integrin-RGD affinity, our results suggest that individual integrin-ligand complexes undergo a marked enhancement in stability when the receptor assembles in the cell membrane. Based on the observation of streptavidin-biotin unbinding, we also conclude that the magnitude of integrin-ligand tension in focal adhesions can reach values that are at least 10 fold larger than was previously estimated using traction force microscopy-based methods.