Biological effects of carbon ion beams with various LETs on budding yeast Saccharomyces cerevisiae.

It has been established that irradiation with higher linear energy transfer (LET) increases lethality and mutagenicity more than that with lower LET. However, the characteristics specific to carbon ion beam have not yet been elucidated. Yeast cells were irradiated with carbon ions with an LET of 13 or 50keV/µm, and cell survival and mutation frequency were analyzed. The results, combined with our previous findings for ions with an LET of 107keV/µm, demonstrated that, in conjunction with an increase in LET, cell survival decreased, while mutation frequency increased. This indicates that a carbon ion beam with a higher LET is more mutagenic than one with a lower LET.

Novel mechanism of regulation of protein 4.1G binding properties through Ca2+/calmodulin-mediated structural changes.

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca(2+)-saturated calmodulin (Ca(2+)/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin-radixin-moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca(2+)/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca(2+)/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca(2+)/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S(71)RGISRFIPPWLKKQKS peptide (pepG) mediates Ca(2+)/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca(2+)/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca(2+)/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.

Human S100A3 tetramerization propagates Ca(2+)/Zn(2+) binding states.

The S100A3 homotetramer assembles upon citrullination of a specific symmetric Arg51 pair on its homodimer interface in human hair cuticular cells. Each S100A3 subunit contains two EF-hand-type Ca(2+)-binding motifs and one (Cys)3His-type Zn(2+)-binding site in the C-terminus. The C-terminal coiled domain is cross-linked to the presumed docking surface of the dimeric S100A3 via a disulfide bridge. The aim of this study was to determine the structural and functional role of the C-terminal Zn(2+)-binding domain, which is unique to S100A3, in homotetramer assembly. The binding of either Ca(2+) or Zn(2+) reduced the α-helix content of S100A3 and modulated its affinity for the other cation. The binding of a single Zn(2+) accelerated the Ca(2+)-dependent tetramerization of S100A3 while inducing an extensive unfolding of helix IV. The Ca(2+) and Zn(2+) binding affinities of S100A3 were enhanced when the other cation bound in concert with the tetramerization of S100A3. Small angle scattering analyses revealed that the overall structure of the S100A3 tetramer bound both Ca(2+) and Zn(2+) had a similar molecular shape to the Ca(2+)-bound form in solution. The binding states of the Ca(2+) or Zn(2+) to each S100A3 subunit within a homotetramer appear to be propagated by sensing the repositioning of helix III and the rearrangement of the C-terminal tail domain. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Fundamental study of the radiation monitoring system based on evaluation of DNA lesions.

The biological dosemeter that measures biological responses to ionising radiation is useful for radiation protection. This paper presents the development and characterisation of a gamma ray irradiation dosimetry system based on real-time PCR (polymerase chain reaction) methodology. Real-time PCR is used to amplify and simultaneously quantify a targeted DNA molecule. If there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment. The essential point of this assay is that DNA lesions caused by ionising radiation block DNA synthesis by DNA polymerase, resulting in a decrease in the amplification of a damaged DNA template compared with that of non-damaged DNA templates.

Flexible structures and ligand interactions of tandem repeats consisting of proline, glycine, asparagine, serine, and/or threonine rich oligopeptides in proteins.

Tandem repeats occur in 14% of all proteins. The repeat unit lengths range from a single amino acid to more than 100 residues and the repeat number is sometimes over 100. Understanding the structures, functions, and evolution of these repeats is a significant goal in both proteomics and genomics. This review summarizes experimental studies addressing structural features of tandem repeats of short oligopeptides that are rich in proline, glycine, asparagine, serine, and/or threonine. The oligopetides include (PGMG) and (PNN) in biomineralization protein (PM27), and (NPNA) in Plasmodium falciparum circumsporozoite protein, (YSPTSPS) in RNA polymerase II, (PHGGGWGQ) in the prion protein, (YGHGGG(N)) and (YNHGGG(G)) in plant glycine-rich proteins, (PGQGQQ), (PGQGQQGQQ) and (GYYPTSOQQ) of wheat HMW glutenin, (FGGMGGGKGG) in Aequipecten abductin. Spectroscopic studies including NMR and CD indicate that these peptides adopt type I and II beta-turns, polyproline II helices, loop conformations, and random coils. Formation of these structures frequently depends on pH, solvent, temperature and hydration. The loop conformations are sometimes stabilized by cation-phi, CH-phi, and/or amino-aromatic interactions. These observations indicate that many tandem repeats are largely flexible. In addition to generating repeating domains and providing flexible linkers between domains, the tandem repeats of (PHGGGWGQ), (YGHGGG(N)) and (YNHGGG(G)) and those in titin bind Cu(2+) ions; whereas, tandem repeats of (NPNA) and those in elastin bind Ca(2+) ions. The interactions of some tandem repeats with various target proteins probably involve an induced fit. The tandem repeats in tropoelastin, flagelliform silk, wheat HMW glutenin, abductin, titin, and human nucleoporin, nup153, are responsible for elastomeric properties.

Solution X-ray scattering reveals a novel structure of calmodulin complexed with a binding domain peptide from the HIV-1 matrix protein p17.

The solution structures of complexes between calcium-saturated calmodulin (Ca (2+)/CaM) and a CaM-binding domain of the HIV-1 matrix protein p17 have been determined by small-angle X-ray scattering with use of synchrotron radiation as an intense and stable X-ray source. We used three synthetic peptides of residues 11-28, 26-47, and 11-47 of p17 to demonstrate the diversity of CaM-binding conformation. Ca (2+)/CaM complexed with residues 11-28 of p17 adopts a dumbbell-like structure at a molar ratio of 1:2, suggesting that the two peptides bind each lobe of CaM, respectively. Ca (2+)/CaM complexed with residues 26-47 of p17 at a molar ratio of 1:1 adopts a globular structure similar to the NMR structure of Ca (2+)/CaM bound to M13, which adopted a compact globular structure. In contrast to these complexes, Ca (2+)/CaM binds directly with both CaM-binding sites of residues 11-47 of p17 at a molar ratio of 1:1, which induces a novel structure different from known structures previously reported between Ca (2+)/CaM and peptide. A tertiary structural model of the novel structure was constructed using the biopolymer module of Insight II 2000 on the basis of the scattering data. The two domains of CaM remain essentially unchanged upon complexation. The hinge motions, however, occur in a highly flexible linker of CaM, in which the electrostatic residues 74Arg, 78Asp, and 82Glu interact with N-terminal electrostatic residues of the peptide (residues 12Glu, 15Arg, and 18Lys). The acidic residues in the N-terminal domain of CaM interact with basic residues in a central part of the peptide, thereby enabling the central part to change the conformations, while an acidic residue in the C-terminal domain interacts with two basic residues in the two helical sites of the peptide. The overall structure of the complex adopts an extended structure with the radius of gyration of 20.5 A and the interdomain distance of 34.2 A. Thus, the complex is principally stabilized by electrostatic interactions. The hydrophobic patches of Ca (2+)/CaM are not responsible for the binding with the hydrophobic residues in the peptide, suggesting that CaM plays a role to sequester the myristic acid moiety of p17.

Target recognition by calmodulin: the role of acid region contiguous to the calmodulin-binding domain of calcineurin A.

Small-angle X-ray scattering was used to investigate the role of acid region contiguous to the calmodulin-binding domain (391-414) of calcineurin in the target recognition by calmodulin. Three synthetic peptides with the residues 385-414, 380-414 and 374-414 of calcineurin A were used for this aim. The X-ray data are consistent with the fact that calmodulin binds all three peptides with or without Ca2+. Without Ca2+, the whole peptide including acid residues interacts with dumbbell shaped calmodulin, while the acid region is extruded from globular shaped calmodulin with Ca2+. Consequently, a conformation of sequence 374-414 in calcineurin might be changed by Ca2+-signal via calmodulin, suggesting the consequence of this region with acid residues in the full activation mechanism of calcineurin by Ca2+-bound calmodulin.

Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src.

pp60v-src tyrosine protein kinase was suggested to interact with Ca2+-bound calmodulin (Ca2+/CaM) through the N-terminal region based on its structural similarities to CAP-23/NAP-22, a myristoylated neuron-specific protein, whose myristoyl group is essential for interaction with Ca2+/CaM; (1) the N terminus of pp60v-src is myristoylated like CAP-23/NAP-22; (2) both lysine residues are required for the myristoylation-dependent interaction and serine residues that are thought to regulate the interaction through the phosphorylations located in the N-terminal region of pp60v-src. To verify this possibility, we investigated the direct interaction between pp60v-src and Ca2+/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60v-src. The binding assay indicated that only the myristoylated peptide binds to Ca2+/CaM, and the non-myristoylated peptide is not able to bind to Ca2+/CaM. Analyses of the binding kinetics revealed two independent reactions with the dissociation constants (KD) of 2.07 x 10(-9)M (KD1) and 3.93 x 10(-6)M (KD2), respectively. Two serine residues near the myristoyl moiety of the peptide (Ser2, Ser11) were phosphorylated by protein kinase C in vitro, and the phosphorylation drastically reduced the interaction. NMR experiments indicated that two molecules of the myristoylated peptide were bound around the hydrophobic clefts of a Ca2+/CaM molecule. The small-angle X-ray scattering analyses showed that the size of the peptide-Ca2+/CaM complex is 2-3A smaller than that of the known Ca2+/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60v-src and Ca2+/CaM in a novel manner different from that of known Ca2+/CaM, the target molecules, interactions.

Unfolding intermediate of a multidomain protein, calmodulin, in urea as revealed by small-angle X-ray scattering.

The denaturation of calmodulin (CaM) induced by urea has been studied by small-angle X-ray scattering, which is a direct way to evaluate the shape changes in a protein molecule. In the absence of Ca(2+), the radii of gyration (R(g)) of CaM are 20.8+/-0.3 A in the native state and about 34+/-1.0 A in the unfolded state. The transition curve derived from Kratky plots indicates a bimodal transition via a stable unfolding intermediate around 2.5 M urea. In the presence of Ca(2+) and in the presence of both Ca(2+) and a target peptide, the R(g) values are 21.5+/-0.3 and 18.1+/-0.3 A in the native state and 26.7+/-0.4 and 24.9+/-0.4 A at 9 M urea, respectively. The results indicate that a stable unfolding intermediate still persists in 9 M urea. The present results suggest that the shape of unfolding intermediates is an asymmetric dumbbell-like structure, one in the folded and one in the unfolded state.

Solution X-ray scattering data show structural differences among chimeras of yeast and chicken calmodulin: implications for structure and function.

We present here the first evidence, obtained by the use of small-angle X-ray scattering, of the solution structures of chimeras constructed from yeast (Saccharomyces cerevisiae, Sc) and chicken (Gallus gallus, Gg) calmodulin (CaM). The chimeric proteins used in this study are Sc(1-129)/Gg(130-148), Sc(1-128)/Gg(129-148), Sc(1-87)/Gg(88-148), and Sc(1-72)/Gg(73-148) CaMs, in which Sc(1-)(n)() and Gg(()(n)(+1)-148) descend from yeast and chicken CaM in the chimeric proteins, respectively. Under the Ca(2+)-saturated condition, the solution structure of Sc(1-128)/Gg(129-148) CaM has a dumbbell-like shape which is characteristic of vertebrate-type CaM, while that of Sc(1-129)/Gg(130-148) CaM takes an intermediate structure between the dumbbell-like shape and a compact globular shape. The results provide the direct evidence that the replacement of Asp(129) with Ser(129) induces an interaction between two lobes of Sc(1-129)/Gg(130-148) CaM and brings them close together. It implies that a site interacting with the N-lobe is induced in the C-lobe, although site IV that is unable to bind Ca(2+) hinders the ability of the C-lobe to undergo the conformational change to the full open state. In the presence of both Ca(2+) and a peptide synthesized to mimic the CaM binding domain on myosin light chain kinase, MLCK-22p, the solution structures of these chimeric CaMs take a similar compact globular shape but their interactions are quite different. The solution structure and interactions of Sc(1-72)/Gg(73-148) CaM are similar to those of Sc(1-87)/Gg(88-148) CaM. The structure of Sc(1-87)/Gg(88-148) CaM is similar to that of Sc(1-128)/Gg(129-148) CaM, but their interactions are different. The result indicates that the replacement of Glu(119) with Ala(119) has a critical effect on their interactions. Thus, the functional differences among these chimeric CaMs, which have been reported previously [Nakashima, K., et al. (1996) Biochemistry 35, 5602-5610], have been interpreted on the basis of the structures and interactions.

Protein conformational changes studied by diffusion NMR spectroscopy: application to helix-loop-helix calcium binding proteins.

Pulsed-field gradient (PFG) diffusion NMR spectroscopy studies were conducted with several helix-loop-helix regulatory Ca(2+)-binding proteins to characterize the conformational changes associated with Ca(2+)-saturation and/or binding targets. The calmodulin (CaM) system was used as a basis for evaluation, with similar hydrodynamic radii (R(h)) obtained for apo- and Ca(2+)-CaM, consistent with previously reported R(h) data. In addition, conformational changes associated with CaM binding to target peptides from myosin light chain kinase (MLCK), phosphodiesterase (PDE), and simian immunodeficiency virus (SIV) were accurately determined compared with small-angle X-ray scattering results. Both sets of data demonstrate the well-established collapse of the extended Ca(2+)-CaM molecule into a globular complex upon peptide binding. The R(h) of CaM complexes with target peptides from CaM-dependent protein kinase I (CaMKI) and an N-terminal portion of the SIV peptide (SIV-N), as well as the anticancer drug cisplatin were also determined. The CaMKI complex demonstrates a collapse analogous to that observed for MLCK, PDE, and SIV, while the SIV-N shows only a partial collapse. Interestingly, the covalent CaM-cisplatin complex shows a near complete collapse, not expected from previous studies. The method was extended to related calcium binding proteins to show that the R(h) of calcium and integrin binding protein (CIB), calbrain, and the calcium-binding region from soybean calcium-dependent protein kinase (CDPK) decrease on Ca(2+)-binding to various extents. Heteronuclear NMR spectroscopy suggests that for CIB and calbrain this is likely because of shifting the equilibrium from unfolded to folded conformations, with calbrain forming a dimer structure. These results demonstrate the utility of PFG-diffusion NMR to rapidly and accurately screen for molecular size changes on protein-ligand and protein-protein interactions for this class of proteins.

Nef of HIV-1 interacts directly with calcium-bound calmodulin.

It was recently found that the myristoyl group of CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is essential for the interaction between the protein and Ca(2+)-bound calmodulin (Ca(2+)/CaM). Based on the N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins, including the Nef protein from human immunodeficiency virus (HIV), we proposed a new hypothesis that the protein myristoylation plays important roles in protein-calmodulin interactions. To investigate the possibility of direct interaction between Nef and calmodulin, we performed structural studies of Ca(2+)/CaM in the presence of a myristoylated peptide corresponding to the N-terminal region of Nef. The dissociation constant between Ca(2+)/CaM and the myristoylated Nef peptide was determined to be 13.7 nM by fluorescence spectroscopy analyses. The NMR experiments indicated that the chemical shifts of some residues on and around the hydrophobic clefts of Ca(2+)/CaM changed markedly in the Ca(2+)/CaM-Nef peptide complex with the molar ratio of 1:2. Correspondingly, the radius of gyration determined by the small angle X-ray scattering measurements is 2-3 A smaller that of Ca(2+)/CaM alone. These results demonstrate clearly that Nef interacts directly with Ca(2+)/CaM.