Fluorine labelling for in situ F NMR in oriented systems.

The focus of this project is to take advantage of the large NMR chemical shift anisotropy of F to determine the orientation of fluorine labeled biomolecules in situ in oriented biological systems such as muscle. The difficulty with a single fluorine atom is that the orientation determined from a chemical shift is not singlevalued in the case of a fully anisotropic chemical shift tensor. The utility of a labeling approach with two fluorine labels in a fixed molecular framework where one of the labels has an axially symmetric chemical shift anisotropy such as a CF group and the other has a fully asymmetric chemical shift anisotropy such as 5-fluorotryptophan is evaluated.

Conversion of a Cardiac Muscle Modulator from an Inhibitor to an Activator.

The binding of calcium to cardiac troponin C (cTnC) enhances the binding of troponin I (cTnI) switch region to the regulatory domain of cTnC (cNTnC) and triggers muscle contraction. Several molecules alter the response of the sarcomere by targeting this interface; virtually all have an aromatic core that binds to the hydrophobic pocket of cNTnC and an aliphatic tail that interacts with the switch region of cTnI. W7 has been extensively studied, and the positively charged tail has been shown to be important for its inhibitory action.

Drugging the Sarcomere, a Delicate Balance: Position of N-Terminal Charge of the Inhibitor W7.

W7 is a sarcomere inhibitor that decreases the calcium sensitivity of force development in cardiac muscle. W7 binds to the interface of the regulatory domain of cardiac troponin C (cNTnC) and the switch region of troponin I (cTnI), decreasing the binding of cTnI to cNTnC, presumably by electrostatic repulsion between the -NH group of W7 and basic amino acids in cTnI. W7 analogs with a -CO tail are inactive.

A Potent Fluorescent Reversible-Covalent Inhibitor of Cardiac Muscle Contraction.

Compounds that directly modulate the response of the cardiac sarcomere have potential in the treatment of cardiac disease. While a number of sarcomere activators have been discovered and extensively studied, very few inhibitors have been identified. We report a potent cardiac sarcomere inhibitor, DN-F01, targeting the cardiac muscle thin filament protein troponin complex.

Structural Basis of Activation of Fast Skeletal Muscle.

Troponin regulates the calcium-mediated activation of skeletal muscle. Muscle weakness in diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy occurs from diminished neuromuscular output. The first direct fast skeletal troponin activator, , amplifies the response of muscle to neuromuscular input.

The Role of Electrostatics in the Mechanism of Cardiac Thin Filament Based Sensitizers.

Heart muscle contraction is regulated by calcium binding to cardiac troponin C. This induces troponin I (cTnI) switch region binding to the regulatory domain of troponin C (cNTnC), pulling the cTnI inhibitory region off actin and triggering muscle contraction. Small molecules targeting this cNTnC-cTnI interface have potential in the treatment of heart disease.

Metabolomic study of disease progression in scrapie prion infected mice; validation of a novel method for brain metabolite extraction.

Introduction: Prion disease is a form of neurodegenerative disease caused by the misfolding and aggregation of cellular prion protein (PrP). The neurotoxicity of the misfolded form of prion protein, PrP still remains understudied. Here we try to investigate this issue using a metabolomics approach.

Thioimidate Bond Formation between Cardiac Troponin C and Nitrile-containing Compounds.

We have investigated the mechanism and reactivity of covalent bond formation between cysteine-84 of the regulatory domain of cardiac troponin C and compounds containing a nitrile moiety similar to the calcium sensitizer levosimendan. The results of modifications to the levosimendan framework ranged from a large increase in covalent bond formation to complete inactivity. We present the biological activity of one of the most potent compounds.

Optimizing fluorine labelling for F solid-state NMR in oriented biological systems.

When planning a fluorine labeling strategy for F solid state NMR (ssNMR) studies of the structure and/or mobility of fluorine labeled compounds in situ in an oriented biological system, it is important to characterize the NMR properties of the label. This manuscript focuses on the characterization of a selection of aromatic fluorine compounds in dimyristoylphosphatidylcholine bilayers using F ssNMR from the standpoint of determining the optimum arrangement of fluorine nuclei on a pendant aromatic ring before incorporation into more complex biological systems.

Feasibility of trifluoromethyl TROSY NMR at high magnetic fields.

Insights into the structure and dynamics of large biological systems has been greatly improved by two concurrent NMR approaches: the application of transverse relaxation-optimized spectroscopy (TROSY) techniques in multi-dimensional NMR, especially the methyl-TROSY, and the resurgence of F NMR using trifluoromethyl (CF) probes. Herein we investigate the feasibility of combining these approaches into a trifluoromethyl-TROSY experiment. Using a CF-labelled parvalbumin, we have evaluated the natural abundance C-F correlation spectra and find no indication of a CF TROSY at high magnetic fields.

Nascent β Structure in the Elongated Hydrophobic Region of a Gerstmann-Sträussler-Scheinker PrP Allele.

Prion diseases are neurodegenerative disorders caused by the misfolding of the cellular prion protein (PrP). Gerstmann-Sträussler-Scheinker syndrome is an inherited prion disease with one early-onset allele (HRdup) containing an eight-amino-acid insertion; this LGGLGGYV insert is positioned after valine 129 (human PrP sequence) in a hydrophobic tract in the natively disordered region. Here we have characterized the structure and explored the molecular motions and dynamics of HRdup PrP and a control allele.

The calcium sensitizer drug MCI-154 binds the structural C-terminal domain of cardiac troponin C.

The compound MCI-154 was previously shown to increase the calcium sensitivity of cardiac muscle contraction. Using solution NMR spectroscopy, we demonstrate that MCI-154 interacts with the calcium-sensing subunit of the cardiac troponin complex, cardiac troponin C (cTnC). Surprisingly, however, it binds only to the structural C-terminal domain of cTnC (cCTnC), and not to the regulatory N-terminal domain (cNTnC) that determines the calcium sensitivity of cardiac muscle.

Structural Changes Induced by the Binding of the Calcium Desensitizer W7 to Cardiac Troponin.

Compounds that directly modulate the affinity of the thin filament calcium regulatory proteins in cardiac muscle have potential for treating heart disease. A recent "proof of concept" study showed that the desensitizer W7 can correct hyper-calcium-sensitive sarcomeres from RCM R193H inhibitory subunit troponin I (cTnI) transgenic mice. We have determined the high-resolution nuclear magnetic resonance solution structure of W7 bound to the regulatory domain of calcium binding subunit troponin C (cNTnC)-cTnI cChimera designed to represent the key aspects of the cTnC-cTnI interface.

Diverse residues of intracellular loop 5 of the Na/H exchanger modulate proton sensing, expression, activity and targeting.

The mammalian Na/H exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pH) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops.

Residue-specific mobility changes in soluble oligomers of the prion protein define regions involved in aggregation.

Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of β-sheet rich, insoluble and protease resistant protein deposits (called PrP) that occur throughout the brain. Formation of synthetic or in vitro PrP can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrP.

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ.

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan.

A novel Gerstmann-Sträussler-Scheinker disease mutation defines a precursor for amyloidogenic 8 kDa PrP fragments and reveals N-terminal structural changes shared by other GSS alleles.

To explore pathogenesis in a young Gerstmann-Sträussler-Scheinker Disease (GSS) patient, the corresponding mutation, an eight-residue duplication in the hydrophobic region (HR), was inserted into the wild type mouse PrP gene. Transgenic (Tg) mouse lines expressing this mutation (Tg.HRdup) developed spontaneous neurologic syndromes and brain extracts hastened disease in low-expressor Tg.

Structures reveal details of small molecule binding to cardiac troponin.

In cardiac and skeletal muscle, the troponin complex turns muscle contraction on and off in a calcium-dependent manner. Many small molecules are known to bind to the troponin complex to modulate its calcium binding affinity, and this may be useful in a broad range of conditions in which striated muscle function is compromised, such as congestive heart failure. As a tool for developing drugs specific for the cardiac isoform of troponin, we have designed a chimeric construct (cChimera) consisting of the regulatory N-terminal domain of cardiac troponin C (cNTnC) fused to the switch region of cardiac troponin I (cTnI), mimicking the key binding event that turns on muscle contraction.

Reversible Covalent Binding to Cardiac Troponin C by the Ca-Sensitizer Levosimendan.

The binding of Ca to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca (Ca-sensitizers) have therapeutic promise.

A Common Phenotype Polymorphism in Mammalian Brains Defined by Concomitant Production of Prolactin and Growth Hormone.

Pituitary Prolactin (PRL) and Growth Hormone (GH) are separately controlled and sub-serve different purposes. Surprisingly, we demonstrate that extra-pituitary expression in the adult mammalian central nervous system (CNS) is coordinated at mRNA and protein levels. However this was not a uniform effect within populations, such that wide inter-individual variation was superimposed on coordinate PRL/GH expression.

Probing the mechanism of cardiovascular drugs using a covalent levosimendan analog.

One approach to improve contraction in the failing heart is the administration of calcium (Ca(2+)) sensitizers. Although it is known that levosimendan and other sensitizers bind to troponin C (cTnC), their in vivo mechanism is not fully understood. Based on levosimendan, we designed a covalent Ca(2+) sensitizer (i9) that targets C84 of cTnC and exchanged this complex into cardiac muscle.

The structural and functional effects of the familial hypertrophic cardiomyopathy-linked cardiac troponin C mutation, L29Q.

Familial hypertrophic cardiomyopathy (FHC) is characterized by severe abnormal cardiac muscle growth. The traditional view of disease progression in FHC is that an increase in the Ca(2+)-sensitivity of cardiac muscle contraction ultimately leads to pathogenic myocardial remodeling, though recent studies suggest this may be an oversimplification. For example, FHC may be developed through altered signaling that prevents downstream regulation of contraction.