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Signal recognition particle in chloroplasts (cpSRP) exhibits the unusual ability to bind and target full-length proteins to the thylakoid membrane. Unlike cytosolic SRPs in prokaryotes and eukaryotes, cpSRP lacks an RNA moiety and functions as a heterodimer composed of a conserved 54-kDa guanosine triphosphatase (cpSRP54) and a unique 43-kDa subunit (cpSRP43). Assembly of the cpSRP heterodimer is a prerequisite for post-translational targeting activities and takes place through interactions between chromatin modifier domain 2 (CD2) of cpSRP43 and a unique 10-amino-acid region in cpSRP54 (cpSRP54(pep)). We have used multidimensional NMR spectroscopy and other biophysical methods to examine the assembly and structure of the cpSRP43-cpSRP54 interface. Our data show that CD2 of cpSRP43 binds to cpSRP54(pep) in a 1:1 stoichiometry with an apparent K(d) of approximately 1.06 muM. Steady-state fluorescence and far-UV circular dichroism data suggest that the CD2-cpSRP54(pep) interaction causes significant conformational changes in both CD2 and the peptide. Comparison of the three-dimensional solution structures of CD2 alone and in complex with cpSRP54(pep) shows that significant structural changes are induced in CD2 in order to establish a binding interface contributed mostly by residues in the N-terminal segment of CD2 (Phe5-Val10) and an arginine doublet (Arg536 and Arg537) in the cpSRP54 peptide. Taken together, our results provide new insights into the mechanism of cpSRP assembly and the structural forces that stabilize the functionally critical cpSRP43-cpSRP54 interaction.

S100A13 is a member of the S100 protein family that is involved in the copper-dependent nonclassical secretion of signal peptideless proteins fibroblast growth factor 1 and interleukin 1 lpha. In this study, we investigate the effects of interplay of Cu2+ and Ca2+ on the structure of S100A13 using a variety of biophysical techniques, including multi-dimensional NMR spectroscopy. Results of the isothermal titration calorimetry experiments show that S100A13 can bind independently to both Ca2+ and Cu2+ with almost equal affinity (Kd in the micromolar range). Terbium binding and isothermal titration calorimetry data reveal that two atoms of Cu2+/Ca2+ bind per subunit of S100A13. Results of the thermal denaturation experiments monitored by far-ultraviolet circular dichroism, limited trypsin digestion, and hydrogen-deuterium exchange (using 1H-15N heteronuclear single quantum coherence spectra) reveal that Ca2+ and Cu2+ have opposite effects on the stability of S100A13. Binding of Ca2+ stabilizes the protein, but the stability of the protein is observed to decrease upon binding to Cu2+. 1H-15N chemical shift perturbation experiments indicate that S100A13 can bind simultaneously to both Ca2+ and Cu2+ and the binding of the metal ions is not mutually exclusive. The results of this study suggest that the Cu2+-binding affinity of S100A13 is important for the formation of the FGF-1 homodimer and the subsequent secretion of the signal peptideless growth factor through the nonclassical release pathway.

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.

S100A13 is a homodimeric protein that belongs to the S100 subfamily of EF-hand Ca2+-binding proteins. S100A13 exhibits unique physical and functional properties not observed in other members of the S100 family. S100A13 is crucial for the non-classical export of acidic fibroblast growth factors (FGFs-1), which lack signal peptide at their N-terminal end. In the present study, we report the three-dimensional solution structure of Ca2+-bound S100A13 using a variety of 3D NMR experiments. The structure of S100A13 is globular with four helices and an antiparallel beta-sheet in each subunit. The dimer interface is formed mainly by an antiparallel arrangement of helices H1, H1', H4, and H4'. Isothermal titration calorimetry (ITC) experiments show that S100A13 binds non-cooperatively to four calcium ions. Prominent differences exist between the three-dimensional structures of S100A13 and other S100 proteins. The hydrophobic pocket that largely contributes to protein-protein interactions in other S100 proteins is absent in S100A13. The structure of S100A13 is characterized by a large patch of negatively charged residues flanked by dense cationic clusters contributed largely by the positively charged residues located at the C-terminal end. Results of ITC experiments reveal that S100A13 lacking the C-terminal segment (residues 88-98) fails to bind FGF-1. The three-dimensional structure of S100A13 not only provides useful clues on its role in the non-classical export of signal peptide-less proteins such as FGF-1 but also paves the way for rational design of drugs against FGF-induced tumors.

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.