Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

BACKGROUND:Protein disulfide isomerase (PDI) is a promising target for combating thrombosis. Extensive research over the past decade has identified numerous PDI-targeting compounds. However, limited information exists regarding how these compounds control PDI activity, which complicates further development. OBJECTIVES:To define the mechanism of action of 2 allosteric antithrombotic compounds of therapeutic interest, quercetin-3-O-rutinoside and bepristat-2a. METHODS:A multipronged approach that integrates single-molecule spectroscopy, steady-state kinetics, single-turnover kinetics, and site-specific mutagenesis. RESULTS:PDI is a thiol isomerase consisting of 2 catalytic a domains and 2 inactive b domains arranged in the order a-b-b'-a'. The active sites CGHC are located in the a and a' domains. The binding site of quercetin-3-O-rutinoside and bepristat-2a is in the b' domain. Using a library of 9 Förster resonance energy transfer sensors, we showed that quercetin-3-O-rutinoside and bepristat-2a globally alter PDI structure and dynamics, leading to ligand-specific modifications of its shape and reorientation of the active sites. Combined with enzyme kinetics and mutagenesis of the active sites, Förster resonance energy transfer data reveal that binding of quercetin-3-O-rutinoside results in a twisted enzyme with reduced affinity for the substrate. In contrast, bepristat-2a promotes a more compact conformation of PDI, in which a greater enzymatic activity is achieved by accelerating the nucleophilic step of the a domain, leading to faster formation of the covalent enzyme-substrate complex. CONCLUSION:This work reveals the mechanistic basis underlying PDI regulation by antithrombotic compounds quercetin-3-O-rutinoside and bepristat-2a and points to novel strategies for furthering the development of PDI-targeting compounds into drugs.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

Human protein disulfide isomerase (PDI) is an essential redox-regulated enzyme required for oxidative protein folding. It comprises four thioredoxin domains, two catalytically active (a, a') and two inactive (b, b'), organized to form a flexible abb'a' U-shape. Snapshots of unbound oxidized and reduced PDI have been obtained by X-ray crystallography. Yet, how PDI's structure changes in response to the redox environment and inhibitor binding remains controversial. Here, we used multiparameter confocal single-molecule FRET to track the movements of the two catalytic domains with high temporal resolution. We found that at equilibrium, PDI visits three structurally distinct conformational ensembles, two "open" (O1 and O2) and one "closed" (C). We show that the redox environment dictates the time spent in each ensemble and the rate at which they exchange. While oxidized PDI samples O1, O2, and C more evenly and in a slower fashion, reduced PDI predominantly populates O1 and O2 and exchanges between them more rapidly, on the submillisecond timescale. These findings were not expected based on crystallographic data. Using mutational analyses, we further demonstrate that the R300-W396 cation-π interaction and active site cysteines dictate, in unexpected ways, how the catalytic domains relocate. Finally, we show that irreversible inhibitors targeting the active sites of reduced PDI did not abolish these protein dynamics but rather shifted the equilibrium toward the closed ensemble. This work introduces a new structural framework that challenges current views of PDI dynamics, helps rationalize its multifaceted role in biology, and should be considered when designing PDI-targeted therapeutics.

PTU files (time-correlated single-photon counting data) of freely diffusing human oxidized and reduced PDI wild-type and variants labeled at positions 57/401, 88/401, 57/467, and 88/467 with Atto 550/647N.

Folding of newly synthesized proteins in the endoplasmic reticulum is assisted by several families of enzymes. One such family is the protein disulfide isomerases (PDIs). PDIs are oxidoreductases, capable of forming new disulfide bonds or breaking existing ones. Structural information on PDIs unbound and bound to substrates is highly desirable for developing targeted therapeutics, yet it has been difficult to obtain by using traditional approaches because of their relatively large size and remarkable flexibility. Single-molecule FRET (smFRET) could be a powerful tool to study PDIs' structure and dynamics under conditions relevant to physiology, but its implementation has been hindered by technical challenges of position-specific fluorophore labeling. We have overcome this limitation by site-specifically engineering fluorescent dyes into human PDI, the founding member of the family. Proof-of-concept smFRET measurements of catalytically active PDI demonstrate, for the first time, the feasibility of this approach, expanding the toolkit for structural studies of PDIs.

beta(2)-glycoprotein I (beta(2)GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that beta(2)GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to beta(2)GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate beta(2)GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of beta(2)GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating beta(2)GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.

Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R2 = 0.85) but not with aPT-A (R2 = 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.

Protein disulfide isomerase (PDI) is a ubiquitous redox-regulated enzyme, which interacts with hundreds of client proteins intracellularly and extracellularly. It comprises two redox-sensitive domains, each hosting the conserved catalytic motif CxxC, two redox-insensitive protein-binding domains, and three linkers. Snapshots of oxidized and reduced PDI have been obtained by X-ray crystallography. However, how PDI9s structure dynamically changes in response to the redox microenvironment and ligand binding remain unknown. Here, we used multiparameter confocal single-molecule Forster resonance energy transfer (smFRET) and multiple FRET pairs to track the movements of the two catalytic domains with high temporal resolution. Our studies document that, at equilibrium, PDI visits three structurally distinct conformational ensembles, two open (O1 and O2) and one (C), whose distribution is dictated by the redox environment. Despite undergoing large conformational changes, the ensembles interconvert remarkably fast, on the sub-millisecond timescale, indicative of a shallow free-energy landscape. Using mutational analyses, we further demonstrate that the two active sites are structurally nonequivalent and that ligands targeting the active sites of reduced PDI shift the equilibrium towards closed conformations of the enzyme. This work introduces a new structural framework that helps rationalize the multifaced role of PDI in biology and may assist drug development.

Venous and arterial thromboses in patients suffering from the autoimmune disorder Antiphospholipid Syndrome (APS) are caused by the presence of antiphospholipid antibodies (aPL). Emerging evidence indicates that autoantibodies targeting the epitope R39-R43 in the N-terminal domain, Domain I (DI), of β 2 -glycoprotein I (β 2 GPI) are among the most pathogenic aPL in patients with APS. How such autoantibodies engage β 2 GPI at the molecular level remains incompletely understood. Here, we have used X-ray crystallography, single-molecule FRET, and small-angle X-ray scattering to demonstrate that, in the free form, under physiological pH and salt concentrations, human recombinant β 2 GPI adopts an elongated, flexible conformation in which DI is exposed to the solvent, thus available for autoantibody recognition. Consistent with this structural model, binding and mutagenesis studies revealed that the elongated form interacts with a pathogenic anti-DI antibody in solution, without the need of phospholipids. Furthermore, complex formation was affected neither by the neighboring domains, nor by the presence of the linkers, nor by the glycosylations. Since the pathogenic autoantibody requires residues R39 and R43 for optimal binding, these findings challenge longstanding postulates in the field envisioning β 2 GPI adopting immunologic inert conformations featuring inaccessibility of the epitope R39-R43 in DI and support an alternative model whereby the preferential binding of anti-DI antibodies towards phospholipid-bound β 2 GPI arises from the ability of the pre-existing elongated form to bind to the membranes and then oligomerize, processes that are likely to be supported by protein conformational changes. Interfering with these steps may limit the pathogenic effects of anti-DI antibodies in APS patients. Significance In the autoimmune disorder called Antiphospholipid Syndrome (APS), the presence of autoantibodies targeting the plasma glycoprotein beta-2 glycoprotein I (β 2 GPI) is associated with arterial and venous thrombosis as well as pregnancy complications. Understanding how β 2 GPI becomes immunogenic and how autoantibodies in complex with β 2 GPI cause the blood to clot remains a top priority in the field. By elucidating the structural architecture of β 2 GPI free in solution, our studies challenge longstanding postulates in the field and shed new light on the pathogenic mechanisms of APS that may help the development of new diagnostics and therapeutic approaches.

β2-Glycoprotein I (β2GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound β2GPI to that in solution. β2GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, β2GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant β2GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular or twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound β2GPI arises from the ability of its preexisting J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of β2GPI provides a strategy to block pathogenic aPLs in APS.

Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are often detected in patients with antiphospholipid syndrome (APS), but how aPS/PT engage prothrombin at the molecular level remains unknown. Here, the antigenic determinants of immunoglobulin G aPS/PT were investigated in 24 triple-positive APS patients at high risk of thrombosis by using prothrombin mutants biochemically trapped in closed and open conformations, and relevant fragments spanning the entire length of prothrombin. Two novel unexpected findings emerged from these studies. First, we discovered that some aPS/PT are unique among other anti-prothrombin antibodies insofar as they efficiently recognize prothrombin in solution after a conformational change requiring exposure of fragment-1 to the solvent. Second, we identified and characterized 2 previously unknown subpopulations of aPS/PT, namely type I and type II, which engage fragment-1 of prothrombin at different epitopes and with different mechanisms. Type I target a discontinuous density-dependent epitope, whereas type II engage the C-terminal portion of the Gla-domain, which remains available for binding even when prothrombin is bound to the phospholipids. Based on these findings, APS patients positive for aPS/PT were classified into 2 groups, group A and group B, according to their autoantibody profile. Group A contains mostly type I antibodies whereas group B contains both type I and type II antibodies. In conclusion, this study offers a first encouraging step toward unveiling the heterogeneity of anti-prothrombin antibodies in correlation with thrombosis, shedding new light on the mechanisms of antigen-autoantibody recognition in APS.

Background. Antiphospholipid antibodies (aPL) recognizing an epitope comprising residues R39-R43 in the N-terminal domain, Domain I (DI), of beta-2 glycoprotein I (b2GPI) are considered among the most pathogenic in patients with Antiphospholipid Syndrome (APS). How such autoantibodies engage b2GPI at the molecular level remains incompletely understood. Aim. To better understand how pathogenic anti-DI antibodies engage b2GPI at the molecular level. Results. Under physiological conditions, b2GPI is believed to adopt a closed conformation featuring an intramolecular interaction between DI and DV with amino acids R39 and R43 in DI being masked by DV. This conformation is therefore predicted to be immunologically inert, incapable of reacting against pathogenic anti-DI antibodies. Once bound to the membranes, however, b2GPI is believed to undergo a dramatic conformational change which liberates DI to the solvent. To get a better grasp of the molecular flexibility of b2GPI under conditions relevant to physiology, we expressed and purified fully-glycosylated human recombinant b2GPI (hr-b2GPI) from HEK293 cells at high yield and purity suitable for structural biology and biophysical studies. After native purification, we found that the recombinant protein bound to heparin and negatively charged phospholipids with affinities comparable to those obtained for b2GPI that was purified from plasma using the perchloric acid method (p-b2GPI); hr-b2GPI also displayed similar reactivity against anti-b2GPI immunoglobulin G antibodies that were isolated from 5 APS patients. Surprisingly, hr-b2GPI and p-b2GPI were structurally similar, too. The X-ray crystal structures of hr-b2GPI and p-b2GPI solved at 2.6 and 2.4 Å resolution were superimposable documenting a J-shaped elongated conformation of the molecule in which DI was located > 90 Å away from the C-terminal DV. Both structures were characterized by 22 oxidized cysteine residues forming 11 disulfide bonds, 4 N-glycosylations, and an intact yet flexible phospholipid-binding loop in DV. Since crystallization occurred at high salt concentrations, validation of the crystal structure of hr-b2GPI in solution was obtained by single-molecule Förster Resonance Energy Transfer (smFRET) and small-angle X-ray scattering (SAXS), while surface plasmon resonance (SPR) was used to probe the binding of a recently developed monoclonal anti-DI antibody, i.e., MBBS, to hr-b2GPI and p-b2GPI in solution. In keeping with the X-ray structural data, donor and acceptor fluorophores incorporated at positions 13/312 in DI and DV and 112/312 in DII and DV reported no measurable energy transfer whereas probes located at positions 13/112 in DI and DII displayed very high energy transfer. Likewise, the scattering profiles of the recombinant and plasma purified proteins returned similar hydrodynamic radii characteristic of elongated, flexible protein structures, and not circular. Notably, both hr-b2GPI and p-b2GPI in the elongated conformation were capable of interacting with MBBS without the need of phospholipids, even though addition of negatively charged phospholipids decreased the apparent dissociation affinity constant due to a reduction of the dissociation rate constant and a remarkable time-dependent accumulation of b2GPI onto the lipid surface, suggestive of a phospholipid-induced oligomerization mechanism. Conclusions. This study demonstrates that human b2GPI can adopt an elongated conformation in solution that is primed for phospholipid, heparin, and autoantibodies binding with DI constitutively exposed to the solvent. The fact that phospholipid-bound b2GPI is a better antigen for anti-DI autoantibody under physiological conditions as compared to the elongated form in solution can be explained by the relatively low affinity and bivalency of such autoantibodies that likely recognize a peptide motif pattern rather than a specific sequence of residues. Whether other context-dependent conformational changes occur after binding of the protein to the lipid surface, thus facilitating aPL binding, remain to be established. Since our studies failed to detect the closed form of b2GPI previously documented by electron and atomic force microscopy studies, it is possible that this conformation may arise from chemical and/or posttranslational modifications that occur in vivo while the protein circulates in the plasma. Disclosures No relevant conflicts of interest to declare.

The clotting factor prothrombin exists in equilibrium between closed and open conformations, but the physiological role of these forms remains unclear. As for other allosteric proteins, elucidation of the linkage between molecular transitions and function is facilitated by reagents stabilized in each of the alternative conformations. The open form of prothrombin has been characterized structurally, but little is known about the architecture of the closed form that predominates in solution under physiological conditions. Using X-ray crystallography and single-molecule FRET, we characterize a prothrombin construct locked in the closed conformation through an engineered disulfide bond. The construct: (i) provides structural validation of the intramolecular collapse of kringle-1 onto the protease domain reported recently; (ii) documents the critical role of the linker connecting kringle-1 to kringle-2 in stabilizing the closed form; and (iii) reveals novel mechanisms to shift the equilibrium toward the open conformation. Together with functional studies, our findings define the role of closed and open conformations in the conversion of prothrombin to thrombin and establish a molecular framework for prothrombin activation that rationalizes existing phenotypes associated with prothrombin mutations and points to new strategies for therapeutic intervention.

Coagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life and a key target of anticoagulant therapy. In plasma, prothrombin circulates in two forms at equilibrium, “closed” (~80%) and “open” (~20%), brokered by the flexibility of the linker regions. Its structure remained elusive until recently when our laboratory solved the first X-ray crystal structure of the zymogen locked in the predominant closed form. Because of this technical breakthrough, fascinating aspects of the biology of prothrombin have started to become apparent, and with this, novel and important questions arise. Here, we examine the significance of the “closed”/“open” equilibrium in the context of the mechanism of thrombin generation. Further, we discuss the potential translational opportunities for the development of next-generation anticoagulants that arise from this discovery. By providing a structural overview of each alternative conformation, this minireview also offers a relevant example of modern structural biology and establishes a practical workflow to elucidate the structural features of analogous clotting and complement factors.

The transport of oxygen and other nonelectrolytes across lipid membranes is known to depend on both diffusion and solubility in the bilayer, and to be affected by changes in the physical state and by the lipid composition, especially the content of cholesterol and unsaturated fatty acids. However, it is not known how these factors affect diffusion and solubility separately. Herein we measured the partition coefficient of oxygen in liposome membranes of dilauroyl-, dimiristoyl- and dipalmitoylphosphatidylcholine in buffer at different temperatures using the equilibrium-shift method with electrochemical detection. The apparent diffusion coefficient was measured following the fluorescence quenching of 1-pyrenedodecanoate inserted in the liposome bilayers under the same conditions. The partition coefficient varied with the temperature and the physical state of the membrane, from below 1 in the gel state to above 2.8 in the liquid-crystalline state in DMPC and DPPC membranes. The partition coefficient was directly proportional to the partial molar volume and was then associated to the increase in free-volume in the membrane as a function of temperature. The apparent diffusion coefficients were corrected by the partition coefficients and found to be nearly the same, with a null dependence on viscosity and physical state of the membrane, probably because the pyrene is disturbing the surrounding lipids and thus becoming insensitive to changes in membrane viscosity. Combining our results with those of others, it is apparent that both solubility and diffusion increase when increasing the temperature or when comparing a membrane in the gel to one in the fluid state.

The cardiac myofilament is a protein assembly that enable the heart to undergo alternating periods of contraction and relaxation, the driving force regulated by Ca2+. Troponin, a three-member protein assembly within the myofilament, acts as a Ca-sensitive switch. In this work, we used single molecule FRET technique to monitor whether the Troponin complex functions as a Ca2+ -sensitive regulatory switch . The results show a population of unactivated troponin under saturating Ca2+ conditions. We propose that the population of unactivated troponin comprises a form of cardiac reserve that is regulated by signaling pathways that target the myofilament.

RNA polymerase (RNAP) is a molecular machine that carries out a series of reactions during transcription initiation: (i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex (RPc). (ii) RNAP unwinds ∼13 base pairs of promoter DNA surrounding the transcription start site, forming a single-stranded region ("transcription bubble"), and yielding an RNAP-promoter open complex (RPo). (iii) RNAP begins synthesis of an RNA product as an RNAP-promoter initial transcribing complex (RPitc). (iv) After RNAP synthesizes an RNA product ∼11 nt in length, RNAP breaks its interactions with the promoter, escapes from the promoter, and begins transcription elongation as an RNAP-DNA elongation complex (RDe). It has been known for four decades that the transcription start site can vary over a range of at least 5 bp--comprising the default start site (position +1), downstream-shifted start sites, (positions +2 and +3), and upstream-sifted start sites (positions −2 and −1)--and that the transcription start site can be re-programmed within this range by the use of appropriate ribodinucleotide primers. However, the mechanistic basis of this flexibility in transcription start-site selection has not been known. In this work, we have used magnetic-tweezers single-molecule nanomanipulation to monitor the extent of RNAP-dependent DNA unwinding in transcription initiation complexes containing ribodinucleotide primers that re-program transcription to start at downstream-shifted start sites (positions +2 or +3) or upstream-shifted start sites (positions −2 or −1). The results indicate that re-programming the transcription start site changes the transcription-bubble size: forcing a downstream-shifted start site increases transcription-bubble size, and forcing upstream-shifted start sites decreases transcription-bubble size. The results support a model in which flexibility in transcription start-site selection is a consequence of pre-initiation transcription-bubble expansion ("pre-initiation scrunching") or pre-initiation transcription-bubble contraction ("pre-initiation unscrunching").

We report pulsed interleaved single pair FRET (spFRET) measurements of freely diffusing reconstituted cardiac myofilaments. Under saturating Ca2+ conditions, the measurements reveal a population of unactivated troponin. We suggest that this population of unactivated troponin comprises a form of cardiac reserve that is regulated by signaling pathways that target the myofilament. The results are interpreted in terms of a coarse-grained free energy landscape model of myofilament activation.