Fernando C. Reinach

Regulatory Properties of the NH2- and COOH-terminal Domains of Troponin T ATPase ACTIVATION AND BINDING TO TROPONIN I AND TROPONIN C

The contraction of skeletal muscle is regulated by Ca2+ binding to troponin C, which results in an internal reorganization of the interactions within the troponin-tropomyosin complex. Troponin T is necessary for Ca2+-dependent inhibition and activation of actomyosin. Troponin T consists of an extended NH2-terminal domain that interacts with tropomyosin and a globular COOH-terminal domain that interacts with tropomyosin, troponin I, and troponin C. In this study we used recombinant troponin T and troponin I fragments to delimit further the structural and regulatory interactions with the thin filament. Our results show the following: (i) the NH2-terminal region of troponin T activates the actomyosin ATPase in the presence of tropomyosin; (ii) the interaction of the globular domain of troponin T with the thin filament blocks ATPase activation in the absence of Ca2+; and (iii) the COOH-terminal region of the globular domain anchors the troponin C-troponin I binary complex to troponin T through a direct Ca2+-independent interaction with the NH2-terminal region of troponin I. This interaction is required for Ca2+-dependent activation of the actomyosin ATPase activity. Based on these results we propose a refined model for the troponin complex and its interaction with the thin filament.

Identification and Genomic Characterization of a New Virus (Tymoviridae Family) Associated with Citrus Sudden Death Disease

ABSTRACT Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.

Parallel Measurement of Ca2+ Binding and Fluorescence Emission upon Ca2+ Titration of Recombinant Skeletal Muscle Troponin C MEASUREMENT OF SEQUENTIAL CALCIUM BINDING TO THE REGULATORY SITES

Calcium binding to chicken recombinant skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W), 5-hydroxytryptophan (F29HW), or 7-azatryptophan (F29ZW) at position 29 was measured by flow dialysis and by fluorescence. Comparative analysis of the results allowed us to determine the influence of each amino acid on the calcium binding properties of the N-terminal regulatory domain of the protein. Compared with TnC, the Ca2+ affinity of N-terminal sites was: 1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW, and 3) decreased slightly in F29HW. The Ca2+titration of F29ZW monitored by fluorescence displayed a bimodal curve related to sequential Ca2+ binding to the two N-terminal Ca2+ binding sites. Single and double mutants of TnC, F29W, F29HW, and F29ZW were constructed by replacing aspartate by alanine at position 30 (site I) or 66 (site II) or both. Ca2+ binding data showed that the Asp → Ala mutation at position 30 impairs calcium binding to site I only, whereas the Asp → Ala mutation at position 66 impairs calcium binding to both sites I and II. Furthermore, the Asp → Ala mutation at position 30 eliminates the differences in Ca2+ affinity observed for replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or 7-azatryptophan. We conclude that position 29 influences the affinity of site I and that Ca2+ binding to site I is dependent on the previous binding of metal to site II.

The complete sequence of a chicken-muscle cDNA encoding the acidic ribosomal protein P1

We have isolated and sequenced a complete cDNA encoding the acidic phosphoprotein P1 from chicken. The analysis of the deduced protein sequence and its comparison with the known sequence of P proteins from human, rat and Artemia salina indicates that the central, alanine-rich region of these proteins was probably generated by internal duplications of the gene followed by modifications within each repeat. This observation explains the length heterogeneity and sequence divergence of this particular region when compared with the highly conserved structure of the remaining segments of the protein.

Adapting to change in Latin America

The recent political and economic changes in Latin America provide a challenge to the scientific community. They may be the very stimulus needed to improve the quality of research.

Molecular Dissection of Regulatory Light Chain Function in Vertebrate Smooth Muscle Myosins

The recent elucidation of the crystal structures of the myosin head and regulatory domain allows us to analyze the changes in myosin structure that occur during regulation. The light chains are the regulatory subunits and are bound to the α-helical segment of the heavy chain (regulatory domain), which extends from the motor domain (containing the ATPase and actin-binding sites) to the head-tail junction. We have used a recombinant DNA approach to investigate how the light chains in this location regulate the motor domain. To identify the subdomains/regions important for regulatory function, we have synthesized a series of mutant regulatory light chains (RLCs) and characterized them by their ability to restore Ca2+ regulation to the desensitized scallop myosin test system. Assays on chimeric RLCs composed of subdomains derived from vertebrate smooth muscle and skeletal muscle myosin RLCs demonstrate that the origin of the third subdomain specifies the regulatory capability of the RLC. A series of smooth muscle myosin RLC mutants with deletions in the fourth subdomain show that the C-terminal helix in this subdomain is essential for regulation. Although the skeletal and scallop RLCs need an intact Ca2+/Mg2+-binding site in the first subdomain for function, the smooth muscle RLC behaves differently and does not require divalent metal binding to this site for regulation. These studies demonstrate the regions of the RLC that are important for regulatory function.