ABSTRACT
The efficient assembly of histone complexes and nucleosomes requires the participation of molecular chaperones. Currently, there is a paucity of data on their mechanism of action. We now present the structure of an N-terminal domain of nucleoplasmin (Np-core) at 2.3 A resolution. The Np-core monomer is an eight-stranded beta barrel that fits snugly within a stable pentamer. In the crystal, two pentamers associate to form a decamer. We show that both Np and Np-core are competent to assemble large complexes that contain the four core histones. Further experiments and modeling suggest that these complexes each contain five histone octamers which dock to a central Np decamer. This work has important ramifications for models of histone storage, sperm chromatin decondensation, and nucleosome assembly.
Subject(s)
Histones/metabolism , Nuclear Proteins/chemistry , Nucleosomes/metabolism , Phosphoproteins/chemistry , Protein Structure, Tertiary , Amino Acid Sequence , Animals , Crystallography, X-Ray , Histones/chemistry , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Nucleoplasmins , Protein Binding , Protein Folding , Sequence AlignmentABSTRACT
Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Proto-Oncogene Proteins , Transcription Factors/metabolism , Animals , Calorimetry , Circular Dichroism , Core Binding Factor Alpha 2 Subunit , Core Binding Factor alpha Subunits , DNA/chemistry , DNA/genetics , DNA-Binding Proteins/chemistry , Dimerization , Models, Molecular , Molecular Weight , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Tertiary , Thermodynamics , Transcription Factor AP-2 , Transcription Factors/chemistry , UltracentrifugationABSTRACT
Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBF alpha subunit and a non-DNA binding CBF beta subunit that increases the affinity of CBF alpha for DNA. We have developed a procedure for overexpressing and purifying full-length CBF beta as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBF beta proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBF beta bind to a CBF alpha DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBF beta is a mixed alpha/beta protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy.
Subject(s)
DNA-Binding Proteins/chemistry , Oxidoreductases , Transcription Factors/chemistry , Circular Dichroism , Core Binding Factor alpha Subunits , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/isolation & purification , Dimerization , Escherichia coli , Genetic Vectors , Glutaredoxins , Plasmids , Protein Structure, Secondary , Proteins/chemistry , Proteins/genetics , Recombinant Fusion Proteins/chemistry , Transcription Factor AP-2 , Transcription Factors/biosynthesis , Transcription Factors/isolation & purificationABSTRACT
Parvalbumins are a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF-hand. Silver hake parvalbumin (SHPV) consists of three EF-type helix-loop-helix regions, two of which have the ability to bind calcium. The three helix-loop-helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV-B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a beta-lineage parvalbumin. SHPV-B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33-amino acid EF fragment (residues 76-108), containing one of the calcium ion binding sites in native SHPV-B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion-induced self-association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge-density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer-dimer equilibrium when complexed with La3+.
Subject(s)
Fishes , Helix-Loop-Helix Motifs , Parvalbumins/chemistry , Amino Acid Sequence , Animals , Binding Sites , Centrifugation, Isopycnic , Circular Dichroism , Mass Spectrometry , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemistry , Phylogeny , Sequence Analysis , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
Increasing public awareness and concern over the possible dangers of exposure to toxic chemicals and hazardous wastes has resulted in a variety of lawsuits brought by plaintiffs claiming injury resulting from chemical exposure. The legal system and its traditional approach to tort cases demands that a plaintiff demonstrate that a particular chemical substance was the "cause in fact" of his injury. However, a plaintiff's inability to present credible scientific evidence sufficient to pinpoint conclusively the specific cause of his injury or disease, particularly in cancer cases, leads to defeat in courts of law. This article discusses the existing barriers to plaintiffs' recovery in toxic tort cases and reviews congressional proposals designed to ease plaintiffs' evidentiary burden and increase their chances of prevailing.
