Nerve growth factor: structure/function relationships.

Nerve growth factor (NGF), which has a tertiary structure based on a cluster of 3 cystine disulfides and 2 very extended, but distorted beta-hairpins, is the prototype of a larger family of neurotrophins. Prior to the availability of cloning techniques, the mouse submandibular gland was the richest source of NGF and provided sufficient material to enable its biochemical characterization. It binds as a dimer to at least 2 cell-surface receptor types expressed in a variety of neuronal and non-neuronal cells. Residues involved in these interactions and in the maintenance of tertiary and quaternary structure have been identified by chemical modification and site-directed mutagenesis, and this information can be related to their location in the 3-dimensional structure. For example, interactions between aromatic residues contribute to the stability of the NGF dimer, and specific surface lysine residues participate in receptor contacts. The conclusion from these studies is that receptor interactions involve broad surface regions, which may be composed of residues from both promoters in the dimer.     

Large-scale chromatin structure and function.

Recent results in living cells have now established the existence of levels of chromatin folding above the 30 nm fiber within interphase chromosomes. We discuss the potential functional impact of this large-scale chromatin organization, including its possible role in regulating gene expression.     

Glucoamylase: structure/function relationships, and protein engineering

Glucoamylases are inverting exo-acting starch hydrolases releasing beta-glucose from the non-reducing ends of starch and related substrates. The majority of glucoamylases are multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain by an O-glycosylated linker region. Three-dimensional structures have been determined of free and inhibitor complexed glucoamylases from Aspergillus awamori var. X100, Aspergillus niger, and Saccharomycopsis fibuligera. The catalytic domain folds as a twisted (alpha/alpha)(6)-barrel with a central funnel-shaped active site, while the starch-binding domain folds as an antiparallel beta-barrel and has two binding sites for starch or beta-cyclodextrin. Certain glucoamylases are widely applied industrially in the manufacture of glucose and fructose syrups. For more than a decade mutational investigations of glucoamylase have addressed fundamental structure/function relationships in the binding and catalytic mechanisms. In parallel, issues of relevance for application have been pursued using protein engineering to improve the industrial properties. The present review focuses on recent findings on the catalytic site, mechanism of action, substrate recognition, the linker region, the multidomain architecture, the engineering of specificity and stability, and roles of individual substrate binding subsites.     

The hemoglobin system of the brown moray Gymnothorax unicolor: structure/function relationships.

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion-exchange chromatography. The oxygen-binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5-8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co-operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co-operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co-operativity, and displayed a normal Bohr effect; the addition of chloride increased co-operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino-acid sequences of the alpha and beta chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.     

The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity.

We have compared the chromatin structure in the active and inactive states at loci encoding the major heat shock protein in Drosophila. DNAase I and micrococcal nuclease were used as probes of higher order organization and nucleosomal integrity. Such integrity is gauged here by the characteristic pattern of discrete DNA fragments produced at specific chromosomal loci by nucleolytic cleavage. The specific fragment patterns are visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets, hybridization with 32P-labeled cloned DNA containing the heat shock genes and autoradiography. Using this criterion, a disruption in nucleosomal and possibly in higher order organization are observed as indicated by a relative loss or smearing of the characteristic discrete DNA fragment patterns from the heat shock loci in the active state. The fragment patterns are restored when cells are allowed to recover from heat shock and these loci return to the inactive state.     

Protein phosphatases in chromatin structure and function

Chromatin structure and dynamics are highly controlled and regulated processes that play an essential role in many aspects of cell biology. The chromatin transition stages and the factors that control this process are regulated by post-translation modifications, including phosphorylation. While the role of protein kinases in chromatin dynamics has been quite well studied, the nature and regulation of the counteracting phosphatases represent an emerging field but are still at their infancy. In this review we summarize the current literature on phosphatases involved in the regulation of chromatin structure and dynamics, with emphases on the major knowledge gaps that should require attention and more investigation.     

ATP依赖的染色质改构复合物及其作用机制

染色质高度紧密的折叠阻止了转录因子和辅因子与DNA的结合, 因而通过染色质重塑以解除这样的抑制环境, 对于转录活动的正常进行是至关重要的。目前认为, 染色质重塑至少是通过两种机制来完成的, 一种是通过ATP依赖的染色质改构复合物, 另一种是通过对组蛋白尾部进行共价修饰的组蛋白修饰酶复合物。文章结合近年来的研究进展, 对前者进行染色质重塑的机制及两者在基因转录调控过程中如何相互协作等进行了论述。     

Amphiphilic alpha-helical antimicrobial peptides and their structure/function relationships

To facilitate microbial membrane invasion, amphiphilic alpha-helical antimicrobial peptides (alpha-AMPs) show a spatial segregation of hydrophobic and hydrophilic residues about the alpha-helical long axis. Here we discuss potential mechanisms by which these peptides are able to disrupt membrane structure and the structural characteristics, which are required for function.     

Vasopressin receptors: structure/function relationships and signal transduction in target cells

Vasopressin, a hypothalamic hormone, acts on its target tissues via three different G protein coupled receptors. The vasopressin V1a and V1b receptors, associated to Gq protein and phospholipase C, are responsible for vasoconstriction and regulation of the corticotroph axis respectively. The V2 vasopressin receptor is coupled to Gs protein and adenylyl cyclase and is responsible for water reabsorption in the renal collecting duct. Mutations of the V2 receptor are involved in diabetes insipidus and most of these mutations result in an endoplasmic reticulum (ER) retention of the mutated receptor. With the V1b receptor model, we have identified a proximal sequence of the C-terminal segment, which is crucial for ER export. Mutations in this short domain result in ER accumulation and degradation of the receptor. SSR 149415, a nonpeptide antagonist of V1bR, which is permeable to cell membrane, is able to rescue the mutant phenotype and acts as a pharmacological chaperone.     

Cytochrome b of protozoan mitochondria: relationships between function and structure.

1. The sensitivity of ubiquinol:cytochrome c reductase to its most powerful inhibitors has been characterized in mitochondria from three ciliate and two trypanosome protozoans and compared with that in mitochondria of animals and plants. 2. Mitochondria of ciliates, particularly those of Tetrahymena pyriformis, are resistant to antimycin. 3. Mitochondria of trypanosomes are quite resistant to stigmatellin, as they exhibit a 40-fold higher titer than that in ciliate or animals mitochondria. 4. Both ciliates and trypanosomes are highly resistant to myxothiazol. 5. Correlations have been drawn between the natural resistance of the protozoan mitochondria to antimycin, stigmatellin and myxothiazol and peculiar features in the structure of their apocytochrome b, on the basis of an accurate alignment of the sequences of this protein.     

Incorporation of DUF/FACT into chromatin enhances the accessibility of nucleosomal DNA

DNA unwinding factor (DUF) was discovered as an essential DNA replication factor in Xenopus egg extracts. DUF consists of an HMG protein and a homolog of Cdc68p/Spt16p, and has the capability of unwinding dsDNA. Here we have examined the interaction of DUF with chromatin. DUF was incorporated into chromatin assembled from sperm heads and from plasmid DNA in egg extracts. It was revealed that the chromatin assembled in egg extracts immunodepleted of DUF is less sensitive to micrococcal nuclease (NNase) digestion than that assembled in control extracts, indicating that chromatin containing DUF has more decompact structure than that without DUF. Also we found that DUF has a high affinity for core histones in vitro. We suggest that the function of DUF may be to make the chromatin structure accessible to replication factors.     

The structure of SV 40 chromatin.

Simian virus 40 (SV40) nucleoprotein complexes were studied with the electron microscope. Depending on the isolation procedure, SV40 chromatin has two different conformations: complexes isolated in the presence of 0.15 M NaCl appeared as very compact globular structures, while those isolated in the presence of 0.6 M NaCl had the typical 'beads-on-a-string' appearance of the primary nucleofilament. Concomitant with this structural change was a variation in the histone pattern and sedimentation behaviour of the complexes: with NaCl at 0.15 mol 1(-1) the isolated complexes contained both the nucleosomal histones and histone H1, and sedimented in sucrose gradients at 70S. Increasing the ionic strength to 0.6 M NaCl resulted in the removal of histone H1 from the complexes and in a decrease of the sedimentation coefficient to 40S. DNA relaxing enzyme is associated with the SV40 nucleoprotein complexes. The numbers of superhelical turns in DNA from compact and open types of complexes were found to be the same. Therefore the transition from the condensed to the open structure of viral chromatin does not require a change in the topological winding number of its DNA.