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Structural characterization of amyloid rich in cross-β structures is crucial for unraveling the molecular basis of protein misfolding and amyloid formation associated with a wide range of human disorders. Elucidation of the β-sheet structure in noncrystalline amyloid has, however, remained an enormous challenge. Here we report structural analyses of the β-sheet structure in a full-length transthyretin amyloid using solid-state NMR spectroscopy. Magic-angle-spinning (MAS) solid-state NMR was employed to investigate native-like β-sheet structures in the amyloid state using selective labeling schemes for more efficient solid-state NMR studies. Analyses of extensive long-range (13)C-(13)C correlation MAS spectra obtained with selectively (13)CO- and (13)Cα-labeled TTR reveal that the two main β-structures in the native state, the CBEF and DAGH β-sheets, remain intact after amyloid formation. The tertiary structural information would be of great use for examining the quaternary structure of TTR amyloid.

Synthetic pyrrole-imidazole polyamides are used to demonstrate that DNA binding by human estrogen-related receptor 2 (hERR2) is sensitive to the presence of polyamides in both the upstream minor groove C-terminal extension (CTE) site and the minor groove of the estrogen response element (ERE) half-site. Major groove interactions mediated by either hERR2 or hERalpha can be inhibited by polyamides occupying the opposing minor groove of the ERE.

The transcriptional coactivator protein CBP and its paralog p300 each contain two homologous zinc-containing TAZ (transcriptional adaptor zinc-binding) domains, which constitute the interaction sites for a number of transcription factors. Both the CBP and p300 TAZ domains in the presence of stoichiometric concentrations of Zn(super 2+) adopt a well-defined structure in solution in the absence of binding partners.

Heteronuclear NMR methods are used to probe the loop conformations in solution in complexes of dihydrofolate reductase (DHFR) formed during the catalytic cycle. The active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle are characterized from the NMR data.

An important role in the regulation of the T cell receptor (TCR)-alpha gene enhancer is played by the lymphoid enhancer-binding factor-1 (LEF-1) that is a sequence specific and cell type-specific transcription factor. The combined use of heteronuclear multidimensional NMR and circular dichroism (CD) to characterize the structure of the free high-mobility group domain of LEF-1 and to elucidate the conformational changes and increase in structural order that occur upon binding cognate DNA are described.

Research has been conducted on the unfolded apomyoglobin. The NMR characterization of this apomyoglobin denaturated in urea has been carried out in investigating polypeptide's behavior under denaturing conditions and the details are presented.

Research has been conducted on the apomyoglobin folding pathways and intermediates. The role of the intrinsic helical properties in the apomyoglobin folding process has been examined via the mutant preparation in which Asn132 and Glu136 have been substituted with glycine and the results are discussed.

Research has been conducted on the enzyme catalytic mechanism. The effect of the polypeptide chain's local motion on this mechanism has been investigated via (super)15 N relaxation data measurements for Escherichia coli dihydrofolate reductase in different complexes which represent different stages in the enzyme catalytic cycle.

Research has been conducted on the apomyoglobin that forms a denatured state under low-salt conditions. The characterization of the conformational properties and the polypeptide backbone dynamics of this state via NMR spectroscopy is described.

Research has been conducted on the cellular prion protein. The dynamics of the prion protein fragments from Syrian hamster has been investigated via NMR relaxation measurements.

The dynamics of polypeptide chain near the active site of beta-lactamase may influence the binding of substrates and/or intermediates' stabilization during enzyme catalysis. Marked changes in the backbone and tryptophan indole dynamics are seen in the flap and minor loop of the enzyme in the presence and absence of tight-binding inhibitor, SB225666.

Apomyoglobin mutation, mediated by phenylalanine replacing the distal histidine64, while stabilizing the protein, it increases the overall rate of protein folding. Data further indicate that replacement of the polar histidine with a nonpolar type stabilizes the protein in the early stages of folding.

Research has been conducted on the Wilms' tumor suppressor protein. The effect of the posttranscriptional modification on this protein is described.

Research has been conducted on the apomyoglobin folded state at pH 4. The use of NMR spectroscopy in investigating the structure and dynamics of this intermediate state is discussed.

Ferric nitrosylleghemoglobin and XAS experiments carried out on frozen aqueous solutions of Lb (super)II NO and Lb (super)III NO are discussed. Multiple-scattering analyses of the XAFS data are presented.

Metallo-beta-lactamase from Bacteroides fragilis has a hairpin loop next to the two zinc molecules at the catalytic site. This part of the enzyme may be very flexible, allowing it to bind to a variety of substrates.

The effects of chain length and the presence and absence of specific capping sequences were examined by studying a peptide of 31 residues consisting of the C-terminal sequence of the four-helix bundle protein myohemerythrin from themiste zostericola. Intact capping sequences increase the population of helix in the central portion of the peptide and limits helix formation to this sequence. Increased length and intact capping sequences were shown to be important in protein folding.

A study of the backbone and tryptophan side-chain dynamics of uniformly 15N-labeled E. coli dihydrofolate reductase(DHFR)-folate complex reveals that the observed time-dependent structural changes of the binary complex are related to catalytic properties of the molecule. It is found that the large amplitude backbone motions, on the picosecond and nanosecond time scales, occurs at regions involved in transition-state stabilization and in ligand-dependent conformational change.The DHFR backbone exhibits diverse dynamical features.

NMR time scale studies reveal that protons from Escherichia coli's apo-dihydrofolate reductase (DHFR) react with two specific domains at room temperature. ROESY spectra assign resonances for the residues 9 to 24 that occur near DHFR's loop I which has an approximate conformational interconversion rate of 35 per second. Loop I significantly influences catalysis, indicating that the exchange rate kinetics affects enzyme functions.

The complementary method of H2D NMR involves the use of circular dichroism to structurally characterize a monomeric model peptide, acetyl-WEAQAREALAKEAAARA-amide. Multiconformational equilibrium is present, as suggested by the occurrence of heavily populated nonhelical structured conformers. This is evident from the long-range NOEs between the methyl protons of leucine-9 and backbone and side-chain protons of amino acids, situated at the N-terminus.

A mixing method was used to examine the kinetics of the unfolding transition of French bean apoplastocyanin which has both unfolding and refolding transitions, and it was found to have an exclusive single-state transition. Two transitions with two separate speeds were observed and used for measuring the refolding reaction. An intermediate which has many features present in the molten globule is produced in the retarded folding interaction wherein the amide protons present in the beta-sheets are sheltered from exchange.

Two-dimensional double quantum nuclear magnetic resonance experiments help to examine the site-specific titration curves for 12 histidine residues in carbon monoxy sperm whale myoglobin (mbCO). The midpoints of the titrations of the other four residues exhibit bounds. Although variation in the structures of crystal and solution are disregarded, the electrostatic behavior during titration is revealed, and a two-step titration is involved in the partial unfolding of MbCO.

Circular dichaoism (CD) and nuclear magnetic resonance spectroscopy (NMR) studies of the folding properties of the Mb-G (Myoglobin) and the Mb-H (Myoglobin-H) peptides of the G and H helix segments of sperm whale myoglobin show that Mb-G possesses few helices in aqueous solution, while Mb-H has a significant helix population. NMR of Mb-H also indicates that Mb-H helices are in dynamic equilibrium with unfolded states, the equilibrium being dependent on solution concentrations. The relative helicity at various sites of the Mb-H peptide has also been studied.

A study of peptide fragments of sperm whale myoglobin, corresponding to the region between the G- and H- helices, in water and solvent mixtures of water and trifluoroethanol (TFE) indicates that turn sequences in proteins help in helix termination in TFE during the initial folding stages in the native protein. Mb-GH5 (Myoglobin-GH5), a five residue peptide, and Mb-GH25, a 25 residue peptide, turn conformation in aqueous solutions though there is no helix formation in the regions on either side of the residue sequence. The studies used circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR) techniques.

The synthesis and study of a series of dilsulphide-bridged dimeric peptides with the entire G- and H-helix sequences of sperm whale myoglobin (Mb) analyzed the long-range interactions in protein folding after the formation of the initial secondary structure. CD spectra show that disulphide bridged peptides made of two H-helices (Mb-HssH) and one G-and one H-helix (Mb-GssH) form helices in waste due to intermolecular attraction. But Mb-GH51, a 51 residue peptide, shows no helical structures in water, and forms few helices even on the addition of TFE. The role of the G-H helical hairpin in protein folding is also discussed.

Extensive assignments of the 1H, 15N and 13C resonances of the N-terminal DNA-binding domain of the GAL4 transactivator protein were obtained from two- and three-dimensional heteronuclear NMR experiments and structural studies based on short-, medium- and long-range NOEs, which establish the global fold of the protein. The results indicate that the peptide consists of two short alpha helices. The obtained solution structure is similar to previously determined structures of GAL4.

The backbone and tryptophan side-chain dynamics of thereduced and oxidized form of Escherichia coli thioredoxin were examined. The aim was to find out whether the changes in dynamics of the reduced and oxidizedthioredoxin permits access by the reduced form to structural states which are not available to the oxidized form. These differences lead to different effectson other thioredoxin activities such as induction of viral DNA polymerase and exonuclease. The results showed differences in dynamic mobility in the between the backbone regions of the two thioredoxin forms. The functional implications of these differences were discussed.

The binding interfaces of enzyme IIAglc and HPr of the phosphoenolpyruvate:sugar phosphotransferase system in Bacillus subtilis duringphosphate transfer was characterized. Nuclear magnetic resonance spectrum analysis allowed the identification of the residues that undergo significant chemical shifts upon complex formation. The results suggest that binding of thetwo enzymes involves predominantly hydrophobic surfaces near the active site His15 of HPr and the phosphoryl acceptor His 38 of IIAglc.

A study of the functions of a highly mobile loop in Escherichia coli Dihydrofolate reductase was studied by constructing a mutant using cassette mutagenesis with four residues deleted in the mid-section of the loop and a glycine inserted to seal the gap. This part of the loop, involving residues 16-20, is disordered in X-ray crystal structures of the apoprotein and the NADP+ binary complex, but forms a hairpin turn which folds over the pteridine moiety of folate in the ternary complex and the nicotinamide moiety of NADP+. The mobility of loop 1 may provide a mechanism for recruiting hydrophobic residues to properly align the nicotinamide and pteridine rings for the hydride-transfer process.

Inverse-detected two-dimensional NMR spectroscopy, using nitrogen-15 labels, serves as a tool for analysing the mobility of residues within the Bacillus subtilis glucose permease IIA domain. Such study is vital to understanding the transcriptional regulation function ofthe phosphoenolpyruvate/sugar phosphotransferase system (PTS), of which glucosepermease IIA is the main control protein. According to a model-free mathematical analysis of the data, mobility is concentrated near the terminals and in irregular loops, coils and pleats. Of particular interest is loop P25-D41 which is close to the active site and may have a role in binding to other proteins.

Nuclear magnetic double- and triple-resonance are used to define nearly the whole structure of the 162-residue Bacillus subtilis glucose permease IIA domain, themain control protein of the phosphoenolpyruvate/sugar phosphotransferase system(PTS). By correlating the alpha-hydrogen and alpha-carbon-13 chemical shifts between two adjacent residues, the sequence of alpha-carbon-13 resonances as well as those of the side-chain spin systems are pieced one by one. Reliance onheteronuclear single-bond rather than hydrogen-hydrogen couplings ensures precise measurements for a protein the size of glucose permease IIA.

Immunogenic peptides of the dominant antigenic region in the transmembrane protein of simian immunodeficiency virus (SIV) possess preferences for water solution conformations other than the extended chain form configuration typical of random coil peptides. This was indicated by conformational studies using nuclear magnetic resonance on two synthetic peptides which correspond to the N- and C- terminal halves of the SIV of macaque origin.

The NMR structure of the full-length tumor suppressor p53 transactivation domain (TAD) in complex with the nuclear coactivator binding domain (NCBD) of key cellular proteins CBP is reported. The results suggested that a salt bridge between D49 of p53 and R2105 of NCBD might contribute to the binding specificity of the two subdomain AD1 and AD2 motifs of p53 to a target protein.

A study was conducted to examine the dynamics of the pathogenic mutants (P101L and H186R), protective mutants (Q167R and Q218K), and wild-type mouse PrP(89?230) at pH 5.5 and 3.5. Findings suggested the possibility that the pathogenic or dominant negative mutations exert their effects on non-native intermediate.