No abstract

A short C-terminal fragment of immunoglobulin-binding domain of streptococcal protein G is known to form nativelike beta-hairpin at physiological conditions. To understand the cooperative folding of the short peptide, eight Ala-substituted mutants of the fragment were investigated with respect to their structural stabilities by analyzing temperature dependence of NMR signals. On comparison of the obtained thermodynamic parameters, we found that the nonpolar residues Tyr45 and Phe52 and the polar residues Asp46 and Thr49 are crucial for the beta-hairpin folding. The results suggest a strong interaction between the nonpolar side chains that participates in a putative hydrophobic cluster and that the polar side chains form a fairly rigid conformation around the loop (46-51). We also investigated the complex formation of the mutants with N-terminal fragment at the variety of temperature to get their thermal unfolding profiles and found that the mutations on the residues Asp46 and Thr49 largely destabilized the complexes, while substitution of Asp47 slightly stabilized the complex. From these results, we deduced that both the hydrophobic cluster formation and the rigidity of the loop (46-51) cooperatively stabilize the beta-hairpin structure of the fragment. These interactions which form a stable beta-hairpin may be the initial structural scaffold which is important in the early folding events of the whole domain.

No abstract

Centromere and kinetochore proteins have a pivotal role in centromere structure, kinetochore formation and sister chromatid separation. However, the molecular architecture and the precise dynamic function of the centromere-kinetochore complex during mitosis remain poorly understood. Here we report the isolation and characterization of human CENP-H. Confocal microscopic analyses of HeLa cells with anti-human CENP-H-specific antibody demonstrated that CENP-H colocalizes with inner kinetochore plate proteins CENP-A and CENP-C in both interphase and metaphase. CENP-H was present outside centromeric heterochromatin, where CENP-B is localized, and inside the kinetochore corona, where CENP-E is localized during prometaphase. Furthermore, CENP-H was detected at neocentromeres, but not at inactive centromeres in stable dicentric chromosomes. In vitro binding assays of human CENP-H with centromere-kinetochore proteins suggest that the CENP-H binds to itself and MCAK, but not to CENP-A, CENP-B or CENP-C. CENP-H multimers were observed in cells in which both FLAG-tagged CENP-H and hemagglutinin-tagged CENP-H were expressed. These results suggest that CENP-H multimers localize constitutively to the inner kinetochore plate and play an important fundamental role in organization and function of the active human centromere-kinetochore complex.

Recently, much attention has been focussed on functional roles of neuropeptides in the nervous system, which presumably include their actions as neurotransmitters or neuroregulators. Especially, substance P was almost established as an excitatory neurotransmitter functioning in the spinal cord.l} Moreover, recent studies have revealed numbers of other neuropeptides to be highly localized in the spinal cord.9~ It is reasonable to expect therefore that there are other as yet undiscovered neuropeptides in the spinal cord possessing significant biological activities. Using the conventional gut contracting assay as a tool, we performed a systematic screening study to identify hitherto unknown neuropeptides in spinal cord. We report here the isolation and the primary structure of two novel neuropeptides, named neurokinin a and p, from porcine spinal cords. Materials and methods. Porcine spinal cords were obtained from a local slaughterhouse and stored at -20°C until used. Reverse phase high performance liquid chromatography (HPLC) was carried out using Nucleosil 5C8, Ultrasphere ODS, Zorbax CN or uBondapak. Phenyl. Aqueous acetonitrile containing 0.1 % trifluoroacetic acid was used with a gradient of acetonitrile to elute peptides. An automated fluorescence detection system utilizing fluorescamine was used for monitoring peptides in column effluents.3~ Amino acid analyses were performed after hydrolyzing the peptides at 110°C for 22 hrs in 6 N HC1 containing 4 % thioglycolic acid. Sequence analyses were carried out by manual Edman method4~ and dansyl method.5~ PTH amino acids and dansyl amino acid amides were identified by HPLC.°>' 7 Biological activity of each fraction was assayed by contraction of guinea pig ileum preparation.Results and discussion. Extraction and purification o f the peptides. Porcine spinal cords (10 kg) were homogenized in 301 of

Dnm1p/Vps1p-like protein (DVLP) is a mammalian member of the dynamin GTPase family, which is classified into subfamilies on the basis of the structural similarity. Mammalian dynamins constitute the dynamin subfamily. DVLP belongs to the Vps1 subfamily, which also includes yeast Vps1p and Dnm1p. Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lacks the pleckstrin homology and Pro-rich domains. Dynamin exists as tetramers under physiological salt conditions, whereas under low salt conditions, it can polymerize into spirals that resemble the collar structures seen at the necks of constricted coated pits. In this study, we found that DVLP is also oligomeric, probably tetrameric, under physiological salt conditions and forms sedimentable large aggregates under low salt conditions. The data indicate that neither the pleckstrin homology nor Pro-rich domain is required for the selfassembly. Analyses using the two-hybrid system and coimmunoprecipitation show that the N-terminal region containing the GTPase domain and a domain (DVH1) conserved across members of the dynamin and Vps1 subfamilies, can interact with the C-terminal region containing another conserved domain (DVH2). The data on the interdomain interaction of DVLP is compatible with the previous reports on the interdomain interaction of dynamin. Thus, the self-assembly mechanism of DVLP appears to resemble that of dynamin, suggesting that DVLP may also be involved in the formation of transport vesicles.Dynamin is a high molecular weight GTP-binding protein with an intrinsic GTPase activity. Many proteins that belong to the dynamin family have been identified in a variety of organisms ranging from yeasts to mammals. These proteins have a highly conserved N-terminal GTPase domain of ϳ300 amino acids (for reviews, see Refs. 1-4). This family is divided into subfamilies on the basis of the structural similarity (2). The dynamin subfamily consists of mammalian dynamins I, II, and III, and the Drosophila shibire gene product. A second subfamily consists of Saccharomyces cerevisiae Vps1p and Dnm1p and a recently identified mammalian member designated DVLP 1 (5), dymple (6), DLP1 (dynamin-like protein 1) (7), or DRP1 (dynamin-related protein 1) (8). Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lack the pleckstrin homology and Pro-rich domains, which are involved in interactions with phospholipids and proteins containing the SH3 domain, respectively. Two plant homologs, phragmoplastin and aG68/ADL1, are grouped into another subfamily. Interferon-inducible Mx proteins and yeast Mgm1p constitute the most diverged subfamily.Members of the dynamin and Vps1 subfamilies have been implicated in intracellular vesicular transport. Analyses using cells transfected with dynamin mutants and using Drosophila shibire mutants demonstrated that dynamins are responsible for budding of endocytic vesicles from the plasma membrane (9 -12). It has als...

We examined the complementation of various pairs of fragments derived from the streptococcal protein G BI domain by NMR and CD. Most were not associated; however, one pair of fragments (1-40) and (41-56) interacted sufficiently enough to regenerate a stable 1:1 complex, Ka = 9 x 10 -6 M. A 2D-NMR analysis showed that the structure of the complex resembled that of native domain. Here we discuss the complementation from the viewpoint of the folding pathway of the protein.

Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-. Although mouse embryos homozygous for COX17 disruption die between embryonic days E8.5 and E10, they develop normally until E6.5. This phenotype is strikingly similar to embryos of Ctr1(؊/؊), a cell surface copper transporter, in its lethality around the time of gastrulation. COX17-deficient embryos exhibit severe reductions in CCO activity at E6.5. Succinate dehydrogenase activity and immunoreactivities for anti-COX subunit antibodies were normal in the COX17(؊/؊) embryos, indicating that this defect was not caused by the deficiency of other complexes and/or subunits but was caused by impaired CCO activation by Cox17p. Since other copper chaperone (Atox1 and CCS)-deficient mice show a more moderate defect, the disruption of the COX17 locus causes the expression of only the phenotype of Ctr1(؊/؊). We found that the activity of lactate dehydrogenase was also normal in E6.5 embryos, implying that the activation of CCO by Cox17p may not be essential to the progress of embryogenesis before gastrulation.