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Chloroplasts contain a unique signal recognition particle (cpSRP). Unlike the cytoplasmic forms, the cpSRP lacks RNA but contains a conserved 54-kDa GTPase and a novel 43-kDa subunit (cpSRP43). Recently, three functionally distinct chromodomains (CDs) have been identified in cpSRP43. In the present study, we report the three-dimensional solution structures of the three CDs (CD1, CD2, and CD3) using a variety of triple resonance NMR experiments. The structure of CD1 consists of a triple-stranded beta-sheet segment. The C-terminal helical segment typically found in the nuclear chromodomains is absent in CD1. The secondary structural elements in CD2 and CD3 include a triple-stranded antiparallel beta-sheet and a C-terminal helix. Interestingly, the orientation of the C-terminal helix is significantly different in the structures of CD2 and CD3. Critical comparison of the structures of the chromodomains of cpSRP43 with those found in nuclear chromodomain proteins revealed that the diverse protein-protein interactions mediated by the CDs appear to stem from the differences that exist in the surface charge potentials of each CD. Results of isothermal titration calorimetry experiments confirmed that only CD2 is involved in binding to cpSRP54. The negatively charged C-terminal helix in CD2 possibly plays a crucial role in the cpSRP54-cpSRP43 interaction.

An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, kappa-hefutoxin1 and kappa-hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that kappa-hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any beta-sheets. Based on the presence of the functional diad (Tyr(5)/Lys(19)) at a distance (6.0 +/- 1.0 A) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. kappa-Hefutoxin 1 not only blocks the voltage-gated K(+)-channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of kappa-hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.

In the present study, we demonstrate the thermal induced amyloid formation in a beta-barrel protein, such as the acidic fibroblast growth factor from Notopthalmus viridescens (nFGF-1). Fibril formation in nFGF-1 is observed to occur maximally at 65 degrees C. Electron microscope analysis of the thermal induced fibrils of nFGF-1 shows that they are filamentous with an average diameter of about 20 nm. X-ray diffraction analysis reveals that the thermal induced fibrils of nFGF-1 have a typical "cross-beta" structure with the beta-strands perpendicular to the fibril axis. By using a variety of biophysical techniques including multidimensional NMR, we demonstrate that fibril formation involves the formation of a partially structured intermediate(s) in the thermal unfolding pathway of the protein (nFGF-1). Results of the anilino-8-napthalene sulfonate binding experiments indicate that fibril formation occurs due to the coalescence of the protein (in the intermediate state(s)) through the solvent-exposed non-polar surface(s). In this study, we also demonstrate that organic osmolytes, such as proline, can efficiently prevent the thermal induced amyloid formation in nFGF-1. Proline is found to stabilize the native conformation of the protein. The addition, proline is observed to increase the cooperativity of the unfolding (native <--> denatured) reaction and consequently decrease the population of the "sticky" thermal equilibrium intermediate(s) responsible for the fibril formation.