Conformational transitions of human calcitonin~hCT! during fibril formation in the acidic and neutral conditions were investigated by high-resolution solid-state 13 C NMR spectroscopy. In aqueous acetic acid solution~pH 3.3!, a local a-helical form is present around Gly10, whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13 C chemical shifts. On the other hand, a local b-sheet form as viewed from Gly10 and Phe22, and both b-sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from a-helix to b-sheet, and from random coil to b-sheet forms occurred in the central and C-terminus regions, respectively, during the fibril formation. The increased 13 C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two-step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local b-sheet conformation at the central region and exhibited a random coil at the C-terminus region. Not only a hydrophobic interaction among the amphiphilic a-helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel b-sheets at pH 7.5 and a mixture of antiparallel and parallel b-sheets at pH 3.3.

Doc2 has two C2 domains that interact with Ca2؉ and phospholipid. Munc13 has two C2 domains and one C1 domain that interacts with phorbol ester or diacylglycerol (DAG) and phospholipid. Both Doc2 and Munc13 are implicated in Ca 2؉ -dependent neurotransmitter release, but their modes of action still remain unclear. We show here that Doc2 interacts with Munc13 both in a cell-free system and in intact PC12 cells during the high K We have isolated Doc2 as a novel protein having two C2 domains that interact with Ca 2ϩ and PL 1 (1). Doc2 consists of two isoforms, Doc2␣ and Doc2␤ (1, 2). Doc2␣ is specifically expressed in neuronal cells, whereas Doc2␤ is ubiquitously expressed (1-3). Both isoforms have at least the N-terminal Doc2-specific region and C-terminal two C2 domains. We have moreover shown that overexpression of the N-terminal fragment of Doc2␣ or its C-terminal fragment including the C2 domains in PC12 cells inhibits Ca 2ϩ -dependent exocytosis (4).These results suggest that Doc2␣ is involved in Ca 2ϩ -dependent exocytosis and interacts with another component of Ca 2ϩ -dependent exocytotic machinery. To clarify the mode of action of Doc2␣ in Ca 2ϩ -dependent exocytosis, it is important to isolate its interacting protein(s). We have attempted here to isolate a Doc2␣-interacting protein from a rat brain cDNA library by use of the yeast two-hybrid system and isolated Munc13 as a Doc2␣-interacting protein.Munc13 has been isolated as a mammalian homologue of Caenorhabditis elegans unc-13, which is implicated in Ca 2ϩ -dependent neurotransmitter release (5, 6). Munc13 has three isoforms, Munc13-1, -2, and -3. All the isoforms have two C2 domains and Munc13-1 has another atypical C2 domain. They have moreover one C1 domain that interacts with PE or DAG and PL (5-7). Munc13 is specifically expressed in neuronal cells, and Munc13-1 is localized at the presynaptic plasma membrane (6).We describe here that Doc2␣ directly interacts with Munc13-1 in a DAG-dependent manner and that the Doc2␣-Munc13-1 interactions play an important role in Ca 2ϩ -dependent exocytotic machinery. EXPERIMENTAL PROCEDURESTwo-hybrid Assay-The N-terminal fragment (1-90 aa) of human Doc2␣ cDNA (1) was inserted into the pBTM116 (pLexA-Doc2␣N). The yeast reporter strain L40 was transformed with pLexA-Doc2␣N and a rat brain cDNA library constructed in pGAD10 (CLONTECH). Library plasmids from positive clones were analyzed by transformation tests and DNA sequencing. Overlapping clones containing the full-length coding region of Munc13-1 were isolated by screening the rat brain cDNA library. The cDNA fragments encoding several Munc13-1 deletion mutants were constructed from the overlapping clones and inserted into pGAD424. The cDNA fragments encoding several Doc2␣ deletion mutants were inserted into pBTM116. After co-transformation into yeast strain L40, ␤-galactosidase activity was assayed by liquid and filter assays (8,9).Preparation of Recombinant Proteins-The cDNA fragments encoding the N-terminal fragment (1-90 aa) of human Doc2␣ (1) and Munc13-1-...

The conformation and dynamics of melittin bound to the dimyristoylphosphatidylcholine (DMPC) bilayer and the magnetic orientation in the lipid bilayer systems were investigated by solid-state (31)P and (13)C NMR spectroscopy. Using (31)P NMR, it was found that melittin-lipid bilayers form magnetically oriented elongated vesicles with the long axis parallel to the magnetic field above the liquid crystalline-gel phase transition temperature (T(m) = 24 degrees C). The conformation, orientation, and dynamics of melittin bound to the membrane were further determined by using this magnetically oriented lipid bilayer system. For this purpose, the (13)C NMR spectra of site-specifically (13)C-labeled melittin bound to the membrane in the static, fast magic angle spinning (MAS) and slow MAS conditions were measured. Subsequently, we analyzed the (13)C chemical shift tensors of carbonyl carbons in the peptide backbone under the conditions where they form an alpha-helix and reorient rapidly about the average helical axis. Finally, it was found that melittin adopts a transmembrane alpha-helix whose average axis is parallel to the bilayer normal. The kink angle between the N- and C-terminal helical rods of melittin in the lipid bilayer is approximately 140 degrees or approximately 160 degrees, which is larger than the value of 120 degrees determined by x-ray diffraction studies. Pore formation was clearly observed below the T(m) in the initial stage of lysis by microscope. This is considered to be caused by the association of melittin molecules in the lipid bilayer.

Solid-state 31P- and 13C-NMR spectra were recorded in melittin-lecithin vesicles composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Highly ordered magnetic alignments were achieved with the membrane surface parallel to the magnetic field above the gel-to-liquid crystalline phase transition temperature (Tc). Using these magnetically oriented vesicle systems, dynamic structures of melittin bound to the vesicles were investigated by analyzing the 13C anisotropic and isotropic chemical shifts of selectively 13C-labeled carbonyl carbons of melittin under the static and magic-angle spinning conditions. These results indicate that melittin molecules adopt an alpha-helical structure and laterally diffuse to rotate rapidly around the membrane normal with tilt angles of the N-terminal helix being -33 degrees and -36 degrees and those of the C-terminal helix being 21 degrees and 25 degrees for DLPC and DPPC vesicles, respectively. The rotational-echo double-resonance method was used to measure the interatomic distance between [1-13C]Val8 and [15N]Leu13 to further identify the bending alpha-helical structure of melittin to possess the interhelical angles of 126 degrees and 119 degrees in DLPC and DPPC membranes, respectively. These analyses further lead to the conclusion that the alpha-helices of melittin molecules penetrate the hydrophobic cores of the bilayers incompletely as a pseudo-trans-membrane structure and induce fusion and disruption of vesicles.

Fibril formation in human calcitonin (hCT) from aqueous solution at pH 4.1 was examined and compared with those at pH 3.3 and 7.5 corresponding to three different net charges by means of site-directed (13)C solid-state NMR spectroscopy. Notably, the observed (13)C chemical shifts and lineshapes of the (13)C CP/MAS spectra differed substantially among fibrils prepared at different pHs. It was found that antiparallel beta-sheet structures were formed at pH 7.5 and 4.1 in the central core regions. In the C-terminal region, random coils were formed at both pH 7.5 and 4.1, although the random coil region at pH 4.1 was larger than that at pH 7.5. Fibrillation kinetics analyzed by a two-step autocatalytic reaction mechanism showed that the rate constants k(1) and k(2) for nucleation and maturation reactions of the fibril formation, respectively, were separately determined and the values correlated well with the net positive charges of Lys(18) and His(20) rather than the existence of a negative charge of Asp(15). Further, an attempt was made to assess interatomic distances between amide nitrogen and carbonyl carbon of neighboring chains of (13)C, (15)N-labeled hCT and a model pentapeptide by (13)C REDOR measurements by taking into account its dipolar interaction analyzed by the 3 spin system proposed previously. A unique chain packing of the antiparallel beta-sheets was proposed as a dominant fibril structure, although the possibility of a contribution of chain packing consisting of sliding one or two residues perpendicular to the fibril direction cannot be ruled out. In addition, it appears that the phenyl rings of Phe(16) are aligned on the same side of the beta-sheet and make the beta-sheet stable by forming pi-pi interactions between the beta-strands.