Proteins can exist in a trinity of structures: the ordered state, the molten globule and the random coil. Five examples follow which suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. 1. In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state. 2. Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle. We propose that the increased charge imbalance promotes its conversion to a much less rigid form. 3. Clusterin contains an ordered domain and also a native molten globular region. The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris. 4. In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity.Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding. 5. Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding about 50 ions/molecule. Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure. In addition to these examples, 16 more proteins with native disorder will be discussed. These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks and entropic bristles.Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence.The sequence/structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank. Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30 % of eucaryotic proteins having disordered regions of length = 50 consecutive residues.

The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51-90), and a DNA dodecamer containing the PRDII site of the interferon-beta promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contracts. The absence of one of these modules in the third DNA binding domain accounts for its-100 fold reduced affinity relative to the second one.

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Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH 2 -dependent monooxygenase (TftD) and an NADH:FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol (2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH 2 . We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 Å resolution, respectively. The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide:isoalloxazine rings in TftC and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was also similar to the known structures of FADH 2 -dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-Di-CHQ. An H289A mutation resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP. Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH 2 is generated by TftC, and TftD uses FADH 2 to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH 2 between TftC and TftD without direct physical interaction between the two enzymes.

Transcription of the gene encoding a 35,000-molecular-weight protein (35K protein) from the EcoRI-S region (86.8 to 87.8 map units) of Autographa california nuclear polyhedrosis virus (AcMNPV) occurs early in infection and declines later. The region promoting the gene for the 35K protein, extending from 426 base pairs (bp) upstream to 12 bp downstream from the RNA start site, was linked to the bacterial chloramphenicol acetyltransferase gene (CAT) for analysis. CAT expression was monitored in cells that were transfected with plasmids containing the promoter-CAT fusion as well as cells infected with recombinant viruses containing the chimeric gene inserted into the AcMNPV genome. Mapping of the 5' ends of CAT-specific RNAs indicated that transcription initiated from the proper sites in both assays; moreover, the promoter fragment retained its early activity, despite an alternate location in the viral genome. The 5' boundary of upstream regulatory sequences was determined by constructing deletions of the promoter fragment extending toward the early RNA start site (position +1). In transient assays, a gradual reduction in CAT expression occurred as sequences from positions -426 to -31 were removed. In contrast, promoter deletions from positions -426 to -155 in recombinant viruses exhibited no effect on CAT expression, whereas deletions to position -55 abolished early expression but had no effect on late expression. Late CAT expression was eliminated when deletions to position -4 removed part of the late RNA start site. DNA signals potentiating early transcription were therefore located upstream (between positions -155 and -55) from those involved in late transcription of the gene encoding the 35K protein. Potential consensus sequences for early and late regulatory elements were identified.

The high-mobility group I(Y) (HMGI(Y)) family of proteins plays an important architectural role in chromatin and have been implicated in the control of inducible gene expression. We have previously shown that expression of HMGI antisense RNA in Jurkat T cells inhibits the activity of the IL-2 promoter. Here we have investigated the role of HMGI(Y) in controlling IL-2 promoter-reporter constructs as well as the endogenous IL-2 gene in both Jurkat T cells and human PBL. We found that the IL-2 promoter has numerous binding sites for HMGI(Y), which overlap or are adjacent to the known transcription factor binding sites. HMGI(Y) modulates binding to the IL-2 promoter of at least three transcription factor families, AP-1, NF-AT and NF-κB. By using a mutant HMGI that cannot bind to DNA but can still interact with the transcription factors, we found that DNA binding by HMGI was not essential for the promotion of transcription factor binding. However, the non-DNA binding mutant acts as a dominant negative protein in transfection assays, suggesting that the formation of functional HMGI(Y)-containing complexes requires DNA binding as well as protein:protein interactions. The alteration of HMGI(Y) levels affects IL-2 promoter activity not only in Jurkat T cells but also in PBL. Importantly, we also show here that expression of the endogenous IL-2 gene as well as proliferation of PBL are affected by changes in HMGI(Y) levels. These results demonstrate a major role for HMGI(Y) in IL-2 expression and hence T cell proliferation.

Campylobacter jejuni is a leading bacterial cause of human gastrointestinal disease worldwide. While C. jejuni is a commensal organism in chickens, case-studies have demonstrated a link between infection with C. jejuni and the consumption of foods that have been cross-contaminated with raw or undercooked poultry. We hypothesized that vaccination of chickens with C. jejuni surface-exposed colonization proteins (SECPs) would reduce the ability of C. jejuni to colonize chickens, thereby reducing the contamination of poultry products at the retail level and potentially providing a safer food product for consumers. To test our hypothesis, we injected chickens with recombinant C. jejuni peptides from CadF, FlaA, FlpA, CmeC, and a CadF-FlaA-FlpA fusion protein. Seven days following challenge, chickens were necropsied and cecal contents were serially diluted and plated to determine the number of C. jejuni per gram of material. The sera from the chickens were also analyzed to determine the concentration and specificity of antibodies reactive against the C. jejuni SECPs. Vaccination of chickens with the CadF, FlaA, and FlpA peptides resulted in a reduction in the number of C. jejuni in the ceca compared to the non-vaccinated C. jejuni-challenged group. The greatest reduction in C. jejuni colonization was observed in chickens injected with the FlaA, FlpA, or CadF-FlaA-FlpA fusion proteins. Vaccination of chickens with different SECPs resulted in the production of C. jejuni-specific IgY antibodies. In summary, we show that the vaccination of poultry with individual C. jejuni SECPs or a combination of SECPs provides protection of chickens from C. jejuni colonization.

Mammalian high-mobility group I nonhistone protein (HMG-I) is a DNA-binding chromatin protein that has been demonstrated both in vitro and in vivo to be localized to the A+T-rich sequences of DNA. Recently an unusual binding domain peptide, "the A-T-hook" motif, that mediates specific interaction of H1MG-I with the minor groove of DNA in vitro has been described. Inspection of the A-T-hook region of the binding domain showed that it matches the consensus sequence for phosphorylation by cdc2 kinase. Here we demonstrate that HMG-I is a substrate for phosphorylation by purified mammalian cdc2 kinase in vitro. The site of phosphorylation by this enzyme is a threonine residue at the amino-terminal end of the principal binding-domain region of the protein. Labeling of mitotically blocked mouse cells with [32P]phosphate demonstrates that this same threonine residue in H1MG-I is also preferentially phosphorylated in vivo. Competition binding studies show that cdc2 phosphorylation of a synthetic bindingdomain peptide significantly weakens its interaction with A+T-rich DNA in vitro, and a similar weakening of DNA binding has been observed for intact murine HMG-I protein phosphorylated by the kinase in vitro. These fndings indicate that cdc2 phosphorylation may significantly alter the DNA-binding properties of the HMG-I proteins. Because many cdc2 substrates are DNA-binding proteins, these results further suggest that alteration of the DNA-binding affinity of a variety of proteins is an important general component of the mechanism by which cdc2 kinase regulates cell cycle progression.The mammalian nonhistone high-mobility group proteins (HMGs) are a relatively abundant class of nuclear proteins thought to be important structural components affecting the conformation and function of chromatin (1). HMG-I and HMG-Y are isoform members of a subgroup of these nonhistone proteins (collectively called the HMG-I family) that are most likely produced by translation of mRNAs derived from a common precursor by alternative splicing (2, 3). The HMG-I proteins (not to be confused with the unrelated HMG-1 chromatin protein; ref.