Chemokine receptors are of great interest as they play a critical role in many immunological and pathological processes. The ability to study chemokine receptors in aqueous solution without detergent would be significant because natural receptors require detergents to become soluble. We previously reported using the QTY code to design detergent-free chemokine receptors. We here report the design of 2 detergent-free chimeric chemokine receptors that were experimentally unattainable in detergent solution. We designed chimeric receptors by switching the N terminus and 3 extracellular (EC) loops between different receptors. Specifically, we replaced the N terminus and 3 EC loops of CCR5QTYwith the N terminus and 3 EC loops of CXCR4. The ligand for CXCR4; namely CXCL12, binds to the chimeric receptor CCR5QTY(7TM)-CXCR4 (N terminus+3 EC loops), but with lower affinity compared to CXCR4; the CCL5 ligand of CCR5 binds the chimeric receptor with ∼20× lower affinity. The chimeric design helps to elucidate the mechanism of native receptor-ligand interaction. We also show that all detergent-free QTY-designed chemokine receptors, expressed inEscherichia coli, bind to their respective chemokines with affinities in the nanomolar (nM) range, similar to the affinities of native receptors and SF9-produced QTY variants. These QTY-designed receptors exhibit remarkable thermostability in the presence of arginine and retain ligand-binding activity after heat treatment at 60 °C for 4 h and 24 h, and at 100 °C for 10 min. Our design approach enables affordable scale-up production of detergent-free QTY variant chemokine receptors with tunable functionality for various uses.

Our results suggest faulty mitophagy in dystrophic hearts due to defects in the PINK1/PARKIN pathway.

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Striated muscle contraction is regulated by dynamic and cooperative interactions among Ca2+, troponin, and tropomyosin on the thin filament. While Ca2+ regulation has been extensively studied, little is known about the dynamics of individual regulatory units and structural changes of individual tropomyosin molecules in relation to their mechanical properties, and how these factors are altered by cardiomyopathy mutations in the Ca2+ regulatory proteins. In this hypothesis paper, we explore how various experimental and analytical approaches could broaden our understanding of the cooperative regulation of cardiac contraction in health and disease.

Cardiac troponin C (cTnC) is a key effector in cardiac muscle excitation-contraction coupling as the Ca2+ sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP) and an acceptor fluorescent protein (YFP). The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC’s N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+. The sensor that exhibited the largest responses in end point titrations, CTV-TnC, (Cerulean, TnC, and Venus) was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca2+-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca2+ versus Mg2+) conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg2+ binding. In conclusion, we successfully generated a novel FRET-Ca2+ sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.

Our studies suggest that downregulation of miR-448-3p leads to the increase in the expression of Ncf1 gene and p47(phox) protein, as well as to the substantial increase in NOX2-derived ROS production. Cellular oxidative stress subsequently triggers events that finally culminate in cardiac tissue damage and development of cardiomyopathy.

Actomyosin kinetics in both skinned skeletal muscle fibers at maximum Ca2+-activation and unregulated in vitro motility assays are modulated by solvent microviscosity in a manner consistent with a diffusion limited process. Viscosity might also influence cardiac thin filament Ca2+-regulatory protein dynamics. In vitro motility assays were conducted using thin filaments reconstituted with recombinant human cardiac troponin and tropomyosin; solvent microviscosity was varied by addition of sucrose or glucose. At saturating Ca2+, filament sliding speed (s) was inversely proportional to viscosity. Ca2+-sensitivity (pCa50) of s decreased markedly with elevated viscosity (η/η0 ≥ ~1.3). For comparison with unloaded motility assays, steady-state isometric force (F) and kinetics of isometric tension redevelopment (kTR) were measured in single, permeabilized porcine cardiomyocytes when viscosity surrounding the myofilaments was altered. Maximum Ca2+-activated F changed little for sucrose ≤ 0.3 M (η/η0 ~1.4) or glucose ≤ 0.875 M (η/η0 ~1.66), but decreased at higher concentrations. Sucrose (0.3 M) or glucose (0.875 M) decreased pCa50 for F. kTR at saturating Ca2+ decreased steeply and monotonically with increased viscosity but there was little effect on kTR at sub-maximum Ca2+. Modeling indicates that increased solutes affect dynamics of cardiac muscle Ca2+-regulatory proteins to a much greater extent than actomyosin cross-bridge cycling.

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