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. The structural data thus obtained suggest that HPS1 and HPS2 are precursors of human protamines HP2 and HP3.     

N-Glycans protect proteins from protease digestion through their binding affinities for aromatic amino acid residues

It was previously revealed [Yamaguchi, H., Nishiyama, T., and Uchida, M. (1999) J. Biochem. 126, 261-265] that N-glycans of both the high-mannose and complex types have binding affinity for aromatic amino acid residues. This study shows that free N-glycans protect proteins from protease digestion through their binding affinities for the aromatic amino acid residues exposed on protein molecules. Protease digestion of bovine pancreatic RNase A and bovine a-lactalbumin was depressed in solutions (1 mM or so) of free N-glycans of both the high-mannose and complex types. The increasing order of the protective effects of the N-glycans paralleled that of their affinities for aromatic amino acid residues; and the presence of aromatic amino acids practically abolished the protective effects of the N-glycans. The N-glycans also depressed the protease digestion of metallothionein, an aromatic amino acid-free protein, in agreement with the observation that the N-glycans also interact with the solvent-exposed aromatic amino acid residues of the proteases. Thus it seems probable that the N-glycans protect proteins from protease digestion by steric hindrance attributable to their binding affinity for the solvent-exposed aromatic amino acid residues of both substrate proteins and proteases.     

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.     

The OmpR-family of proteins: insight into the tertiary structure and functions of two-component regulator proteins

The Escherichia coli DNA-binding protein OmpR is the best characterized of those regulator proteins making up "two-component system," the simplest known form of bacterial signal transduction systems. Previous inspections of the E. coli genome DNA sequences have revealed that there are 15 proteins whose amino acid sequences show extensive similarities to that of OmpR (the OmpR-family of proteins). The three-dimensional structures of several OmpR-family proteins have been determined. In this review, we investigated the structures and amino acid sequences of this family of proteins. The results reveal several notable conservative varieties in their tertiary structures and functions.     

The amino-acid sequence of beta-lactoglobulin II from horse colostrum (Equus caballus, Perissodactyla): beta-lactoglobulins are retinol-binding proteins

beta-Lactoglobulin isolated from horse colostrum is heterogeneous and contains two components: beta-lactoglobulin I and beta-lactoglobulin II. These two proteins are monomeric and show differences in their electrophoretic mobilities, chain lengths and primary structures. The complete amino-acid sequence of beta-lactoglobulin II was determined by automated Edman degradation of the intact protein and of the peptides derived from these by digestion with trypsin or chymotrypsin and by chemical cleavage with cyanogen bromide. Unlike other beta-lactoglobulins which contain 162 amino acids, horse beta-lactoglobulin II is unique in that it contains 166 amino acids. The additional four amino acids represent an insertion between positions 116 and 117 of other beta-lactoglobulins so far sequenced, including horse beta-lactoglobulin I. Sequence comparison of beta-lactoglobulins I and II from horse colostrum reveals 48 amino acid substitutions (30%). Such a diversity between members of the beta-lactoglobulin gene family has not been encountered before. Sequence comparison with bovine beta-lactoglobulin A shows 85 amino acid replacements accounting for 53% of the residues. The structural homology with human retinol-binding protein may reveal similar biological functions and clues to the origin of milk proteins.     

Correlation of gene and protein structures in the FXYD family proteins

The FXYD family proteins are auxiliary subunits of the Na,K-ATPase, expressed primarily in tissues that specialize in fluid or solute transport, or that are electrically excitable. These proteins range in size from about 60 to 160 amino acid residues, and share a core homology of 35 amino acid residues in and around a single transmembrane segment. Despite their relatively small sizes, they are all encoded by genes with six to nine small exons. We show that the helical secondary structures of three FXYD family members, FXYD1, FXYD3, and FXYD4, determined in micelles by NMR spectroscopy, reflect the structures of their corresponding genes. The coincidence of helical regions, and connecting segments, with the positions of intron-exon junctions in the genes, support the hypothesis that the FXYD proteins may have been assembled from discrete structural modules through exon shuffling.     

Chemical composition of the gastric shield of a bivalve, Zyrphea crispata, and of the teeth of the gizzard of a gastropod opisthobranch, Aplysia punctata

The chemical composition of the gastric shield of the bivalve Zyrphea crispata is very similar to that of the teeth of the gizzard of the gastropod opisthobranch Aplysia punctata. Both structures are indeed mainly made up of proteins and chitin. Their protein fractions, although heterogeneous, have almost the same amino acid composition. The proportion of free chitin is remarkably high, amounting to 74–78% of the total chitin. These cuticular structures certainly play a similar function in the gastric digestion, according to the presence of some hydrolases mainly laminarinase, the activity of which is much higher than in the crystalline style. Despite their morphological dissimilarity and specialization in two distinct classes of mollusca, the gastric shield and the teeth of the gizzard thus appear to be homologous structures on the basis of their chemical composition.     

Localization of the amino-acid exchange in protein S5 from an Escherichia coli mutant resistant to spectinomycin

Summary Ribosomal proteins S5 were isolated from E. coli B wild type and from a spectinomycin resistant mutant derived from it. After tryptic digestion the peptides were isolated and their amino acid compositions compared. An amino acid replacement, namely arginine by leucine, was found at the C-terminus of peptide T8. This result, together with our previous studies, shows that in spectinomycin resistant mutants the amino acid replacements are clustered within a very narrow region of protein S5.     

Second codon positions of genes and the secondary structures of proteins. Relationships and implications for the origin of the genetic code

The nucleotide frequencies in the second codon positions of genes are remarkably different for the coding regions that correspond to different secondary structures in the encoded proteins, namely, helix, beta-strand and aperiodic structures. Indeed, hydrophobic and hydrophilic amino acids are encoded by codons having U or A, respectively, in their second position. Moreover, the beta-strand structure is strongly hydrophobic, while aperiodic structures contain more hydrophilic amino acids. The relationship between nucleotide frequencies and protein secondary structures is associated not only with the physico-chemical properties of these structures but also with the organisation of the genetic code. In fact, this organisation seems to have evolved so as to preserve the secondary structures of proteins by preventing deleterious amino acid substitutions that could modify the physico-chemical properties required for an optimal structure.     

In situ proteolytic digestion of inclusion body polypeptides occurs as a cascade process

Misfolded proteins undergo a preferent degradation ruled by the housekeeping bacterial proteolytic system, but upon precipitation as inclusion bodies their stability dramatically increases. The susceptibility of aggregated polypeptides to proteolytic attack remains essentially unexplored in bacteria and also in eukaryotic cells. We have studied here the in vitro proteolysis of beta-galactosidase fusion proteins by trypsin treatment of purified inclusion bodies. A cascade digestion process similar to that occurring in vivo has been observed in the insoluble fraction of the digestion reaction. This suggests that major protease target sites are not either lost or newly generated by protein precipitation and that the digestion occurs in situ probably on solvent-exposed surfaces of inclusion bodies. In addition, the sequence of the proteolytic attack is influenced by protein determinants other than amino acid sequence, the early digestion steps having a dramatic influence on the further cleavage susceptibility of the intermediate degradation fragments. These observations indicate unexpected conformational changes of inclusion body proteins during their site-limited digestion, that could promote protein release from aggregates, thus partially accounting for the plasticity of in vivo protein precipitation and solubilization in bacteria.     

Distribution of amino acid residues and residue-residue contacts in molecular chaperones.

The amino acid distribution and residue-residue contacts in molecular chaperones are different when compared to normal globular proteins. The study of molecular chaperones reveals a different surrounding environment to exist for the residues Cys, Trp, and His which may play an important role in determining the chaperone structures. Unlike globular proteins, it has been observed that a one-to-one correspondence between the amino acid distribution in a sequence and the structures of molecular chaperones. The preference of amino acid residues surrounding all 20 types of residues in secondary structures and their accessible surface areas have been analysed.     

beta-Granins: 21 kDa co-secreted peptides of the insulin granule closely related to adrenal medullary chromogranin A

Three closely related forms of a 21 kDa protein which is co-secreted with insulin have been purified and analysed. These differed in behaviour on ion-exchange chromatography but were indistinguishable by their susceptibility to staphylococcal V8 proteinase digestion, amino acid composition or N-terminal amino acid sequence. Their amino acid composition and N-terminal sequences were remarkably similar to adrenal medullary chromogranin A, a much larger protein (72 kDa). Antibodies to chromogranin A also reacted strongly with the 21 kDa protein in isolated insulin granules. It is concluded that the 21 kDa proteins either represent a repeated domain within the chromogranin molecule or a closely related gene product. The name beta-granin is proposed for these proteins.     

Structural identity between the iron- and manganese-containing superoxide dismutases

We have recently reported the first complete amino acid sequence of an iron-containing superoxide dismutase. The iron enzyme is thought to be closely homologous to the manganese-containing superoxide dismutases. The availability of complete amino acid sequence information for four manganese superoxide dismutases and the crystal structures for two iron and two manganese superoxide dismutases prompted us to investigate the degree of homology between the two proteins at various levels. We report that it is not possible to clearly distinguish the two proteins on the basis of their secondary or tertiary structures. It would appear that a small number of single site substitutions are responsible for conferring distinguishing properties between the two proteins. Substitution of glycine 77 and glutamine 154 by a glutamine and an alanine respectively in Photobacterium leiognathi iron superoxide dismutase may distinguish the kinetic and other particular properties of this protein from the manganese protein (and other iron superoxide dismutases). Furthermore the primary structure of both the iron and manganese proteins does not appear to have any homology with any other known amino acid sequence.     

Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents: the yeast salinity stress response

Protein expression profiles in yeast cells, in response to salinity stress, were determined using the cleavable isotope-coded affinity tag (cICAT) labeling strategy. The analysis included separation of the mixed protein samples by SDS-PAGE, followed by excision of the entire gel lane, and division of the lane into 14 gel regions. Regions were subjected to in-gel digestion, biotin affinity chromatography, and analysis by nano-scale microcapillary liquid chromatography coupled to tandem mass spectrometry. The novel (13)C-labeled ICAT reagents have identical elution profiles for labeled peptide pairs and broadly spread the distribution of labeled peptides during reversed-phase chromatography. A total of 560 proteins were identified and quantified, with 51 displaying more than 2-fold expression differences. In addition to some known proteins involved in salt stress, four RNA-binding proteins were found to be up-regulated by high salinity, suggesting that selective RNA export from the nucleus is important for the salt-stress response. Some proteins involved in amino acid synthesis, which have been observed to be up-regulated by amino acid starvation, were also found to increase their abundance on salt stress. These results indicate that salt stress and amino acid starvation cause overlapping cellular responses and are likely to be physiologically linked.     

Two-dimensional NMR approaches to the study of protein structure and function

Two-dimensional Fourier transform methods for homonuclear proton NMR spectroscopy have been introduced by Wüthrich and Ernst as a means of extending assignments in spectra of proteins. Multinuclear two-dimensional approaches also appear promising. We are applying current one- and two-dimensional NMR methods to protein family members that differ from one another by one or more amino acid substitutions. The overall goal is to elucidate relationships among the sequences, structures, and functions of these proteins: for example, to delineate primary structural requirements for changes in observable properties such as conformation, amino acid side chain dynamics, hydrogen exchange dynamics, pK'a values, and oxidation-reduction potentials. The ovomucoids from a variety of species of birds, which include a single set of 12 pairs of third-domain proteins (Mr = 6062 for turkey third domain, similar for others) that differ by single amino acid substitutions, provide a favorable system for the study of the structural and dynamic effects of single amino acid replacements. X-ray crystallographic structures of two ovomucoid third domains are available. Other series of proteins being studied by these methods include the photosynthetic electron transport proteins ferredoxin and plastocyanin.     

Biochemical properties of the major proteins from Rhodnius prolixus eggshell

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis.     

A simple method for displaying the hydropathic character of a protein

A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 20 amino acid side-chains is taken into consideration. The scale is based on an amalgam of experimental observations derived from the literature. The program uses a moving-segment approach that continuously determines the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus. At the same time, a midpoint line is printed that corresponds to the grand average of the hydropathy of the amino acid compositions found in most of the sequenced proteins. In the case of soluble, globular proteins there is a remarkable correspondence between the interior portions of their sequence and the regions appearing on the hydrophobic side of the midpoint line, as well as the exterior portions and the regions on the hydrophilic side. The correlation was demonstrated by comparisons between the plotted values and known structures determined by crystallography. In the case of membrane-bound proteins, the portions of their sequences that are located within the lipid bilayer are also clearly delineated by large uninterrupted areas on the hydrophobic side of the midpoint line. As such, the membrane-spanning segments of these proteins can be identified by this procedure. Although the method is not unique and embodies principles that have long been appreciated, its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.     

Proteins of the Bacillus stearothermophilus ribosome. The amino acid sequences of proteins S5 and L30

The amino acid sequences of ribosomal proteins S5 and L30 from Bacillus stearothermophilus have been determined. These proteins have recently been crystallized in our institute. Sequence data were obtained by manual sequencing of peptides derived from cyanogen bromide cleavage and digestion with trypsin and chymotrypsin or thermolysin. Proteins S5 and L30 contain 166 and 62 amino acid residues and have calculated Mr values of 17,628 and 7,053, respectively. Comparison of the sequences with those of the homologous proteins from Escherichia coli shows 55% identical residues for S5 and 53% for L30. For both proteins, the distribution of conserved and substituted regions is not uniform throughout the molecule. Secondary structure predictions were carried out for the B. stearothermophilus proteins. Comparison with the results for the homologous E. coli proteins indicated similar secondary structural order for the molecules from the two species.