Critical role of the H6-H7 loop in the conformational adaptation of all-trans retinoic acid and synthetic retinoids within the ligand-binding site of RARalpha.

The pleiotropic effects of the natural and synthetic retinoids are mediated by the activation of the two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoic X receptors (RXRs). At the molecular level, these events begin with the specific ligand recognition by a nuclear receptor subtype. The adaptation of ligands to the receptor binding site leads to an optimal number of interactions for binding and selectivity which justifies elucidation of the structural requirements of the ligand binding pocket.

Critical role of tyrosine 277 in the ligand-binding and transactivating properties of retinoic acid receptor alpha.

Retinoic acid receptors specifically bind all-trans-retinoic acid (RA) and function as RA-inducible transcriptional regulatory factors. Binding of RA to RARalpha, beta, and gamma is sensitive to nitration with tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that are important for RA binding, we carried out chemical modification experiments with purified RARalpha ligand-binding domain (RARalpha-LBD) subjected to partial acid hydrolysis and selective proteolysis.

The DNA-binding protein II from Zymomonas mobilis. Complete amino acid sequence and interaction with DNA.

The primary structure of the DNA-binding protein II from Zymomonas mobilis has been determined from data provided by automated Edman degradation of the intact protein and of peptides derived from cleavage at aspartic acid and arginine residues. When compared with the homologous protein isolated from other bacteria, the DNA-binding protein II from Z mobilis shows many substitutions. Several non-conservative substitutions at positions usually highly conserved in this type of protein probably account for the weaker DNA-binding activity of this protein compared to that of the E coli protein.

Successive elution by ion-exchange chromatography of H3-H4 histone complexes differing in their degree of acetylation.

On Biorex 70 ion exchanger at neutral pH the histones H3 and H4 are usually eluted by 4 M guanidinium chloride (gdm Cl). In order to protect cysteines and methionines from oxidation we systematically added 2-mercaptoethanol to the elution buffer. This resulted in the two histones being unexpectedly eluted together at around 1 M gdm Cl.

Application of electrospray and fast atom bombardment mass spectrometry to the identification of post-translational and other chemical modifications of proteins and peptides.

Mass spectrometry is a very powerful tool in the identification of chemical modifications of proteins and peptides. Often these modifications cannot be determined by conventional techniques. This report describes the combined use of electrospray ionization mass spectrometry and fast atom bombardment mass spectrometry to complete the primary structure of proteins and peptides.

Amino acid sequence of the human intermediate basic protein 2 (HPI2) from sperm nuclei. Structural relationship with protamine P2.

Human intermediate basic protein 2 (HPI2) is a low-molecular-mass basic protein present in small amounts in human sperm nuclei. The amino acid composition of the protein, its N-terminal amino acid sequence and peptide maps obtained after digestion with endoproteinases Lys-C and Glu-C, reveal that HPI2 is structurally related to human protamine species P2 (HP2), which is rich in Arg, His and Cys residues. Compared to HP2, which is one of the two major sperm protamines, HPI2 has an N-terminal extension of 24 residues which includes six acidic residues and does not possess any Arg residues.

A corrected primary structure for dog-fish Scylliorhinus caniculus protamine Z3.

We have redetermined the primary structure for dog-fish protamine using automated amino-acid sequencing associated to mass spectrometry techniques and report, on the basis of these findings, that the previously published amino-acid sequence is incorrect. The correct protamine sequence is 37 amino acids long and differs from the original published sequence by the C-terminal hexapeptide Arg32-Gly-Arg-Arg-Ser-Arg37.

Determination of the DNA-interacting region of the archaebacterial chromosomal protein MC1. Photocrosslinks with 5-bromouracil-substituted DNA.

Protein MC1 is the major chromosomal protein in methanosarcinaceae. Using photochemical crosslinking on 5-bromouracil-substituted DNA, we identified the region of the protein that interacts with it. This region is located in the C-terminal part of the polypeptide chain, and the crosslinked amino-acids are in the region 74-86.

Complete sequence of Sipunculus nudus erythrocyte histone H2B and its gene. Identification of an N,N-dimethylproline residue at the amino-terminus.

The complete amino acid sequence (122 residues) of histone H2B from erythrocytes of the marine worm Sipunculus nudus, has been established from sequence analysis of peptides generated by highly specific cleavage of the protein and from the nucleotide sequence of the encoding gene. The isolation of the H2B gene was facilitated by using a highly specific nucleotide probe, devised from amino acids 58-68 of the protein. The presence of an N,N-dimethylproline residue at the amino-terminus of the protein was established from data provided by mass spectrometry and NMR spectroscopy.

Cuttlefish sperm protamines. 1. Amino acid sequences of two distinct variants.

The amino acid sequences of two cuttlefish protamine variants Sp1 and Sp2 have been established from automated sequence analysis and mass spectrometry data. Sp1 (57 residues) and Sp2 (56 residues) have molecular masses of 8410 and 8253 Da, respectively. They are almost identical proteins which differ only by one residue of arginine and the position of two of the serine residues (14 and 37 in Sp1; 13 and 35 in Sp2).

Primary structure of the chromosomal proteins MC1a, MC1b, and MC1c from the archaebacterium Methanothrix soehngenii.

The chromosomal protein MC1 of Methanothrix soehngenii is a family of three variants a, b, and c. These are small basic polypeptides of 89, 87, and 90 residues, respectively. Their primary structures have been determined from automated sequence analyses of the intact proteins and from structural data provided by peptides derived from the variants by cleavage at aspartic acid, glutamic acid, arginine, and methionine residues.

Primary structure of the chromosomal protein MC1 from the archaebacterium Methanosarcina sp. CHTI 55.

The DNA of the thermophilic archaebacterium Methanosarcina sp. CHTI 55 has been shown to be associated with two proteins called MC1 and MC2, of molecular mass 11 kDa and 17 kDa (Chartier et al. (1988) Biochim.

'In situ' Edman degradation of protein(s) blotted to immobilon membranes suitable for unblocking reversible chemical modification and elimination of coomassie blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Conditions for unblocking reversible chemical modifications such as maleylation or citraconylation 'in situ' at the N-terminus of proteins after transfer of proteins to immobilon membranes from SDS-PAGE are described. Demaleylation or decitraconylation occurred at 55 degrees C in 70% formic acid (pH 1.50) during 60 min.

Comparison of the amino acid sequences of human protamines HP2 and HP3 and of intermediate basic nuclear proteins HPS1 and HPS2. Structural evidence that HPS1 and HPS2 are pro-protamines.

Two intermediate nuclear basic proteins HPS1 and HPS2 were isolated from human sperm. They were characterized by their electrophoretic mobility in acid-urea gels, their amino acid composition, and their peptide maps after digestion by endoproteinase Lys-C and by endoproteinase Glu-C. Their amino-terminal amino acid sequences have also been determined.

Isolation and characterization of ATP-dependent proteolytically active ubiquitin in cock testis.

1. We have successfully isolated and purified ubiquitin from cock testis by using an inhibitor, p-CMB (p-chloromercuribenzoate), which is one of the inhibitors specific for thiol-proteases and with the following procedures: heating up to 85 degrees C, ammonium sulfate fractionation, gel filtration on Sephadex G-75, chromatography on DE-52 and CM-11 and lyophilization. 2.

Isolation and characterization of two protamines St1 and St2 from stallion spermatozoa, and amino-acid sequence of the major protamine St1.

Two protamines, St1 and St2, were isolated from stallion sperm nuclei, where they represent about 75 and 25%, respectively, of the total basic protein complement. The primary structure of protamine St1 (49 residues; Mr approximately equal to 6600) has been determined. The structure of this protamine is compared to the amino-acid sequence of other mammalian protamines already known.

Purification and characterization of nuclear basic proteins of human sperm.

Highly purified nuclei were obtained from human sperm without protein loss through the use of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), a newly available detergent. The basic protein complement of these nuclei is highly heterogeneous and comprises histones (some of which are testis-specific), protamines and proteins of intermediate basicity and molecular size. The protamines belong to two different classes of protein.

Primary structure of the two variants of a sperm-specific histone H1 from the annelid Platynereis dumerilii.

The amino acid sequences of the two variants (H1a 121 residues and H1b 119 residues) of the sperm-specific histone H1 from the polychaete annelid Platynereis dumerilii have been completely established. Comparison of the sequences of these two variants shows one deletion of two residues in histone H1b and 22 substitents, of which most occur in the globular domain. The two variants differ highly in a sequence of nine residues adjacent to the conservative phenylalanine residue of histone H1 (64-72 in H1a, 62-70 in H1b) which makes H1a less hydrophobic than H1b.

Primary structure of the ram (Ovis aries) protamine.

The amino acid sequence of the protamine isolated from mature sperm nuclei of the ram (Ovis aries) has been established from automated sequence analysis of the S-carboxymethylated protamine. Ram and bull protamines differ only by two point changes and the deletion in bull protamine of the tripeptide Cys39-Arg-Arg41. In mammalian protamines the central region (residues 13-36) consisting mainly of arginine clusters appears to be conserved whereas the N-terminal and C-terminal regions are more variable.

Primary structure of histone H2A from nucleated erythrocyte of the marine worm Sipunculus nudus. Presence of two forms of H2A in the sipunculid chromatin.

The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with alpha-chymotrypsin and from structural studies of tryptic peptides of the protein. By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non-conservative.

Primary structure of the DNA-binding protein HRm from Rhizobium meliloti.

The amino acid sequence of protein HRm, a DNA-binding HU-type protein of 90 residues (Mr 9303), isolated from Rhizobium meliloti, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at arginine and aspartic acid residues. The comparison of the primary structure of protein HRm with that of other HU-type proteins shows that two short sequences, of 7 and 6 residues respectively, located in the median part of the molecule, appear highly conserved and may be important in the function of the protein.

Primary structure of histone H2A from gonads of the starfish Asterias rubens.

The complete amino acid sequence (124 residues) of histone H2A from gonads of the starfish Asterias rubens has been established from automated sequence analyses of large fragments obtained by staphylococcal protease digestion of histone H2A and by limited hydrolysis of H2A-H2B complex with mouse submaxillary gland protease and from structural studies of peptides generated by enzymatic hydrolyses of these fragments or of the protein. By comparison with calf homologous histone, the starfish histone H2A shows 5 deletions and 12 substitutions. Half of the substitutions are non-conservative.