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We determined the three-dimensional structure of the PHD finger of the rice Siz/PIAS-type SUMO ligase, OsSiz1, by NMR spectroscopy and investigated binding ability for a variety of methylated histone H3 tails, showing that OsSiz1–PHD primarily recognizes dimethylated Arg2 of the histone H3 and that methylations at Arg2 and Lys4 reveal synergy effect on binding to OsSiz1–PHD. The K4 cage of OsSiz1–PHD for trimethylated Lys4 of H3K4me3 was similar to that of the BPTF–PHD finger, while the R2 pocket for Arg2 was different. It is intriguing that the PHD module of Siz/PIAS plays an important role, with collaboration with the DNA binding domain SAP, in gene regulation through SUMOylation of a variety of effectors associated with the methylated arginine-riched chromatin domains.

The monomeric Alzheimer's beta amyloid peptide, Abeta, is known to adopt a disordered state in water at room temperature, and a circular dichroism (CD) spectroscopy experiment has provided the secondary-structure contents for the disordered state: 70% random, 25% beta-structural, and 5% helical. We performed an enhanced conformational sampling (multicanonical molecular dynamics simulation) of a 25-residue segment (residues 12-36) of Abeta in explicit water and obtained the conformational ensemble over a wide temperature range. The secondary-structure contents calculated from the conformational ensemble at 300 degrees K reproduced the experimental secondary-structure contents. The constructed free-energy landscape at 300 degrees K was not plain but rugged with five clearly distinguishable clusters, and each cluster had its own characteristic tertiary structure: a helix-structural cluster, two beta-structural clusters, and two random-structural clusters. This indicates that the contribution from the five individual clusters determines the secondary-structure contents experimentally measured. The helical cluster had a similarity with a stable helical structure for monomeric Abeta in 2,2,2-trifluoroethanol (TFE)/water determined by an NMR experiment: The positions of helices in the helical cluster were the same as those in the NMR structure, and the residue-residue contact patterns were also similar with those of the NMR structure. The cluster-cluster separation in the conformational space indicates that free-energy barriers separate the clusters at 300 degrees K. The two beta-structural clusters were characterized by different strand-strand hydrogen-bond (H-bond) patterns, suggesting that the free-energy barrier between the two clusters is due to the H-bond rearrangements.

Novel pulse sequences incorporating the double pulsed field gradient spin-echo technique are presented that have particular use in identifying macromolecular bound water. The use of these sequences is illustrated using ribonuclease T1. Five amide protons cross-relaxing with bound water protons were observed. Examination of the crystal structure revealed that all of these amide protons donate hydrogen bonds or are in close proximity to water molecules with very low temperature factors, indicating that these amide protons are highly correlated with the bound water molecules. This method rapidly provides reliable information for characterizing macromolecular bound water molecules. Copyright © 2002 John Wiley & Sons, Ltd.

The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS(60-137), H-NS(70-137) and H-NS(80-137), whereas it was much weaker for H-NS(91-137). Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS(60-137) forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS(60-137) was nearly identical to that of H-NS(91-137). 1H and 15N chemical shift perturbations induced by complex formation of H-NS(60-137) with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e. residues A80-K96 and T110-A117, play an essential role in DNA binding.

Sunlight was found to strongly induce the formation of Nϵ-(carboxymethyl)lysine (CML) from glycated polylysine in the presence of Fe(III) ion. The initial step of this Fe(III)-catalyzed CML formation was noted to be similar to that of blueprint photography as was confirmed by the production of Turnbull's blue in sunlight-exposed glycated human serum albumin ferricyanide solution in the presence of Fe(III). Based on this, photoinduced oxidative C–C bond cleavage of the Amadori compound was assumed to be initiated by photochemical single electron transfer from ligand to Fe(III) in the Fe(III)–Amadori compound complex affording the Fe(II)–Amadori compound radical intermediate, which eventually yields either CML or active oxygen species. CML is thus a useful oxidative stress marker. The mechanism proposed here would explain the high accumulation of CML in lens protein and skin actinic elastosis.

The 13C nuclear magnetic resonance studies have been carried out on histones H1 and H5, by focusing our interest on possible formation of specific salt bridges between acidic and basic amino acid residues in the proteins and also on the structural difference between the two proteins. The 13C chemical shift and pKa values of the carboxyl group of glutamic acid residues in the histones coincided with those of free glutamic acid. Based on this result and another experiment using completely modified lysine residues in the histones, no evidence for a specific interaction between acidic and basic residues has been found. It has also been shown that the pH-effects of aliphatic and aromatic resonances are quite different between H1 and H5, suggesting that the globular domain of H5 is more stable than that of H1. The correlation time (1.5 ns) for the alpha-carbons of H5 estimated from 13C nuclear Overhauser enhancement was twice as long as that of H1 (0.9 ns), indicating that the backbone in the N-terminal and C-terminal domains of H5 is less mobile than that of H1.

To obtain more useful information about the effect of the degree of stereoregularity on the motion of the polymer chain, the dielectric and dilatometric measurements were made for a series of stereoregular poly(methyl methacrylates) (PMMA). The α- and β-absorptions were observed in each sample, of which the dielectric behaviors of the α-process are discussed. The temperature dependence of the relaxation time of the α-process was sufficiently represented by the WLF equation and the resulting values of the parameters fg and B in the modified WLF equation were found smaller for isotactic-rich PMMA than those values for syndiotactic PMMA. It may be deduced from these results that the chain mobility of the isotactic PMMA is larger than that of the syndiotactic. The dielectric increment of the α-process in the isotactic PMMA is much larger than that in the syndiotactic PMMA, increasing rapidly with temperature, and taking its maximum in the temperature range of 55 to 60°C. The dielectric transition was clearly observed in the case of isotactic-rich PMMA.

The effects of stereoregularity on the low-temperature relaxation processes were studied by dynamic mechanical measurements on isotactic and syndiotactic polyisobutyl methacrylates (iso-PiBMA and syn-PiBMA). The α, β, and γ relaxation processes were observed in both stereoregular forms. Both the α, and β loss peaks were at lower temperatures for iso-PiBMA than for syn-PiBMA. The γ loss peak was observed at about −155°C at 30 Hz for both forms, and the apparent activation energy of this process was same for both samples within experimental error (6.7 ± 0.5 kcal/mole). It was reduced from these results that the α and β processes are both considerably influenced by the isotactic configuration but the γ process is not.