Tomoko Tada

Molecular cloning in yeast by in vivo homologous recombination of the yeast putative α1 subunit of the voltage-gated calcium channel

Saccharomyces cerevisiae has only one gene encoding a putative voltage‐gated Ca2+ channel pore‐forming subunit, CCH1, which is not possible to be cloned by conventional molecular cloning techniques using Escherichia coli. Here, we report the successful cloning of CCH1 in yeast by in vivo homologous recombination without using E. coli. Overexpression of the cloned CCH1 or MID1 alone, which encodes a putative stretch‐activated Ca2+ channel component, does not increase Ca2+ uptake activity, but co‐overexpression results in a 2‐ to 3‐fold increase. Overexpression of CCH1 does not substantially complement the lethality and low Ca2+ uptake activity of a mid1 mutant and vice versa. These results indicate that co‐overproduction of Cch1 and Mid1 is sufficient to increase Ca2+ uptake activity.

The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys73 and Cys120 in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4–5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.

Preparation of heat-induced artificial collagen gels based on collagen-mimetic dendrimers

Collagen, a major component of the extracellular matrix, contains Gly-Pro-Hyp repeats that form a hydrogel. In this study, artificial collagen-mimetic materials were designed. Synthetic dendritic macromolecules were fully modified with (Pro-Hyp-Gly)n and named collagen-mimetic dendrimers. A collagen-like triple helical structure was observed by circular dichroism spectrometry, with an efficiency that depended on the peptide length. A (Pro-Hyp-Gly)10-modified dendrimer exhibited the most efficient triple helix formation. Thermal stability was enhanced by clustering at the surface of the dendrimer. The (Pro-Hyp-Gly)10-modified dendrimer was assembled by heating and the assembly was affected by temperature, time and concentration. Hydrogels based on the (Pro-Hyp-Gly)10-modified dendrimer, but not on the peptide itself, were successfully prepared by heating. The sol–gel transition behavior was similar to natural collagen but not gelatin, which is thermally denatured collagen. Dynamic rheological analysis showed that the sol–gel transition temperature and the strength depended on the concentration. Thus, the collagen-mimetic dendrimer incorporating (Pro-Hyp-Gly)10 is an injectable and controllable artificial collagen gel.

Temperature-dependent higher order structures of the (Pro-Pro-Gly)10-modified dendrimer†

Collagen is the most abundant protein in mammals and is widely used as a biomaterial for tissue engineering and drug delivery. We previously reported that dendrimers and linear polymers, modified with collagen model peptides (Pro-Pro-Gly)₅, form a collagen-like triple-helical structure; however, its triple helicity needs improvement. In this study, a collagen-mimic dendrimer modified with the longer collagen model peptides, (Pro-Pro-Gly)₁₀, was synthesized and named PPG10-den. Circular dichroism analysis shows that the efficiency of the triple helix formation in PPG10-den was much improved over the original. The X-ray diffraction analysis suggests that the higher order structure was similar to the collagen triple helix. The thermal stability of the triple helix in PPG10-den was higher than in the PPG10 peptide itself and our previous collagen-mimic polymers using (Pro-Pro-Gly)₅. Interestingly, PPG10-den also assembled at low temperatures. Self-assembled structures with spherical and rod-like shapes were observed by transmission electron microscopy. Furthermore, a hydrogel of PPG10-den was successfully prepared which exhibited the sol-gel transition around 45°C. Therefore, the collagen-mimic dendrimer is a potential temperature-dependent biomaterial.

Temperature-sensitive elastin-mimetic dendrimers: Effect of peptide length and dendrimer generation to temperature sensitivity.

Dendrimers are synthetic macromolecules with unique structure, which are a potential scaffold for peptides. Elastin is one of the main components of extracellular matrix and a temperature-sensitive biomacromolecule. Previously, Val-Pro-Gly-Val-Gly peptides have been conjugated to a dendrimer for designing an elastin-mimetic dendrimer. In this study, various elastin-mimetic dendrimers using different length peptides and different dendrimer generations were synthesized to control the temperature dependency. The elastin-mimetic dendrimers formed β-turn structure by heating, which was similar to the elastin-like peptides. The elastin-mimetic dendrimers exhibited an inverse phase transition, largely depending on the peptide length and slightly depending on the dendrimer generation. The elastin-mimetic dendrimers formed aggregates after the phase transition. The endothermal peak was observed in elastin-mimetic dendrimers with long peptides, but not with short ones. The peptide length and the dendrimer generation are important factors to tune the temperature dependency on the elastin-mimetic dendrimer.

Essential, Completely Conserved Glycine Residue in the Domain III S2–S3 Linker of Voltage-gated Calcium Channel α1 Subunits in Yeast and Mammals

Voltage-gated Ca2+ channels (VGCCs) mediate the influx of Ca2+ that regulates many cellular events, and mutations in VGCC genes cause serious hereditary diseases in mammals. The yeast Saccharomyces cerevisiae has only one gene encoding the putative pore-forming α1 subunit of VGCC, CCH1. Here, we identify a cch1 allele producing a completely nonfunctional Cch1 protein with a Gly1265 to Glu substitution present in the domain III S2–S3 cytoplasmic linker. Comparison of amino acid sequences of this linker among 58 VGCC α1 subunits from 17 species reveals that a Gly residue whose position corresponds to that of the Cch1 Gly1265 is completely conserved from yeasts to humans. Systematic amino acid substitution analysis using 10 amino acids with different chemical and structural properties indicates that the Gly1265 is essential for Cch1 function because of the smallest residue volume. Replacement of the Gly959 residue of a rat brain Cav1.2 α1 subunit (rbCII), positionally corresponding to the yeast Cch1 Gly1265, with Glu, Ser, Lys, or Ala results in the loss of Ba2+ currents, as revealed by the patch clamp method. These results suggest that the Gly residue in the domain III S2–S3 linker is functionally indispensable from yeasts to mammals. Because the Gly residue has never been studied in any VGCC, these findings provide new insights into the structure-function relationships of VGCCs.

Interaction of the N‐terminal domain of Escherichia coli heat‐shock protein ClpB and protein aggregates during chaperone activity

The Escherichia coli heat‐shock protein ClpB reactivates protein aggregates in cooperation with the DnaK chaperone system. The ClpB N‐terminal domain plays an important role in the chaperone activity, but its mechanism remains unknown. In this study, we investigated the effect of the ClpB N‐terminal domain on malate dehydrogenase (MDH) refolding. ClpB reduced the yield of MDH refolding by a strong interaction with the intermediate. However, the refolding kinetics was not affected by deletion of the ClpB N‐terminal domain (ClpBΔN), indicating that MDH refolding was affected by interaction with the N‐terminal domain. In addition, the MDH refolding yield increased 50% in the presence of the ClpB N‐terminal fragment (ClpBN). Fluorescence polarization analysis showed that this chaperone‐like activity is explained best by a weak interaction between ClpBN and the reversible aggregate of MDH. The dissociation constant of ClpBN and the reversible aggregate was estimated as 45 μM from the calculation of the refolding kinetics. Amino acid substitutions at Leu 97 and Leu 110 on the ClpBN surface reduced the chaperone‐like activity and the affinity to the substrate. In addition, these residues are involved in stimulation of ATPase activity in ClpB. Thus, Leu 97 and Leu 110 are responsible for the substrate recognition and the regulation of ATP‐induced ClpB conformational change.

Hyperactive and hypoactive mutations in Cch1, a yeast homologue of the voltage-gated calcium-channel pore-forming subunit

The yeast Saccharomyces cerevisiae CCH1 gene encodes a homologue of the pore-forming α1 subunit of mammalian voltage-gated calcium channels. Cch1 cooperates with Mid1, a candidate for a putative, functional homologue of the mammalian regulatory subunit α2/δ, and is essential for Ca(2+) influx induced by several stimuli. Here, we characterized two mutant alleles of CCH1, CCH1* (or CCH1-star, carrying four point mutations: V49A, N1066D, Y1145H and N1330S) and cch1-2 (formerly designated mid3-2). The product of CCH1* displayed a marked increase in Ca(2+) uptake activity in the presence and absence of α-factor, and its increased activity was still dependent on Mid1. Mutations in CCH1* did not affect its susceptibility to regulation by calcineurin. In addition, not only was the N1066D mutation in the cytoplasmic loop between domains II and III responsible for the increased activity of Cch1*, but also substitution of another negatively charged amino acid Glu for Asn(1066) resulted in a significant increase in the Ca(2+) uptake activity of Cch1. This is the first report of a hyperactive mutation in Cch1. On the other hand, the cch1-2 allele possesses the P1228L mutation located in the extracellular S1-S2 linker of domain III. The Pro(1228) residue is highly conserved from fungi to humans, and the P1228L mutation led to a partial loss in Cch1 function, but did not affect the localization and expression of Cch1. The results extend our understanding of the structure-function relationship and functional regulation of Cch1.

Differences in pressure and temperature transitions of proteins and polymer gels

Pressure-driven and temperature-driven transitions of two thermoresponsive polymers, poly(N-isopropylacrylamide) (pNIPAM) and poly(N-vinylisobutyramide) (pNVIBA)), in both a soluble linear polymer form and a cross-linked hydro-gel form, were examined by a dynamic light-scattering method and direct microscopic observation, respectively. Their behavior was compared with that of protein systems. Changes in some characteristic parameters in the time-intensity correlation functions of dynamic light-scattering measurement of aqueous solutions of pNIPAM at various pressures and temperatures showed no essential differences during temperature and pressure scanning and, as a whole, the motions of polymers in aqueous solutions were similar in two types of transitions until chain shrinkage occurred. The gels (cross-linked polymer gels) prepared from the thermoresponsive polymers also showed similar volume transitions responding to the pressure and temperature increase. In temperature transitions, however, gels showed drastic volume shrinkage with loss of transparency, while pressure-induced transition showed a slow recovery of transparency while keeping the size, after first transient drastic volume shrinkage with loss of transparency. At a temperature slightly higher than the transition under atmospheric temperature, so-called reentry of the volume change and recovery of the transparency were observed during the pressure-increasing process, which implies much smaller aggregation or non-aggregated collapsed polymer chains in the gel at higher pressures, indicating a certain mechanistic difference of the dehydration processes induced by temperature and pressure.

Biochemical Characterization of an Adenylate Cyclase, CyaB1, in the Cyanobacterium Anabaena sp. Strain PCC 7120

cyaB1 gene encodes a novel type of adenylate cyclase. The catalytic domain is located in the carboxyl-terminal half, while the GAF and PAS domains are conserved in the amino-terminal half. Recombinant CyaB1 and a truncated CyaB1 lacking the amino-terminal domain (ΔN–CyaB1) were purified and characterized. The purified CyaB1 is activated by divalent cations, such as Mg2+ and Mn2+, like other types of adenylate cyclase. The activity of CyaB1 was slightly elevated by forskolin, but was not affected by cGMP, irrespective of the presence of the cGMP binding motif in the GAF domain. The specific activity of ΔN–CyaB1 is one-eighteenth that of CyaB1, whereas the Km values of both proteins are almost the same. The results suggest that the amino-terminal half has a positive regulatory effect on the catalytic activity.