Structure-based statistical analysis of transmembrane helices

Recent advances in determination of the high-resolution structure of membrane proteins now enable analysis of the main features of amino acids in transmembrane (TM) segments in comparison with amino acids in water-soluble helices. In this work, we conducted a large-scale analysis of the prevalent locations of amino acids by using a data set of 170 structures of integral membrane proteins obtained from the MPtopo database and 930 structures of water-soluble helical proteins obtained from the protein data bank. Large hydrophobic amino acids (Leu, Val, Ile, and Phe) plus Gly were clearly prevalent in TM helices whereas polar amino acids (Glu, Lys, Asp, Arg, and Gln) were less frequent in this type of helix. The distribution of amino acids along TM helices was also examined. As expected, hydrophobic and slightly polar amino acids are commonly found in the hydrophobic core of the membrane whereas aromatic (Trp and Tyr), Pro, and the hydrophilic amino acids (Asn, His, and Gln) occur more frequently in the interface regions. Charged amino acids are also statistically prevalent outside the hydrophobic core of the membrane, and whereas acidic amino acids are frequently found at both cytoplasmic and extra-cytoplasmic interfaces, basic amino acids cluster at the cytoplasmic interface. These results strongly support the experimentally demonstrated biased distribution of positively charged amino acids (that is, the so-called the positive-inside rule) with structural data.     

The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition

Glycosylphosphatidylinositol-anchored influenza hemagglutinin (GPI-HA) mediates hemifusion, whereas chimeras with foreign transmembrane (TM) domains mediate full fusion. A possible explanation for these observations is that the TM domain must be a critical length in order for HA to promote full fusion. To test this hypothesis, we analyzed biochemical properties and fusion phenotypes of HA with alterations in its 27-amino acid TM domain. Our mutants included sequential 2-amino acid (Delta2-Delta14) and an 11-amino acid deletion from the COOH-terminal end, deletions of 6 or 8 amino acids from the NH(2)-terminal and middle regions, and a deletion of 12 amino acids from the NH(2)-terminal end of the TM domain. We also made several point mutations in the TM domain. All of the mutants except Delta14 were expressed at the cell surface and displayed biochemical properties virtually identical to wild-type HA. All the mutants that were expressed at the cell surface promoted full fusion, with the notable exception of deletions of >10 amino acids. A mutant in which 11 amino acids were deleted was severely impaired in promoting full fusion. Mutants in which 12 amino acids were deleted (from either end) mediated only hemifusion. Hence, a TM domain of 17 amino acids is needed to efficiently promote full fusion. Addition of either the hydrophilic HA cytoplasmic tail sequence or a single arginine to Delta12 HA, the hemifusion mutant that terminates with 15 (hydrophobic) amino acids of the HA TM domain, restored full fusion activity. Our data support a model in which the TM domain must span the bilayer to promote full fusion.     

Isolation of Antigenically Active Components from Leptospiral Serovar-Specific Lipopoly-saccharide Antigen by Alkaline Treatment

The serovar-specific main antigen (TM antigen) of Leptospira interrogans serovar canicola, which has lipopolysaccharide properties, was treated with 0.1 n sodium hydroxide. This treatment degraded the antigen into two major antigenic components, one of high and one of low molecular weight. The component with the lower molecular weight (approximately 4,000 daltons) consisted mainly of carbohydrates, having lost almost all of the fatty acid and protein components of the original antigen. Although the substance lacked immunoprecipitable activity, it continued to show serovar-specific inhibitory potency in a radioimmunoassay system as well as in a microscopic immunoagglutination reaction of the organisms. The antigenic activity of the compound was also reduced by periodate oxidation as was that of the TM antigen. A component with the same chemical and physicochemical properties was also produced by alkaline treatment from a different serotype TM antigen (serovar kremastos Kyoto), but it showed no antigenic activity.     

Multivariate design and evaluation of a set of RGRPQ-derived innate immunity peptides

Oral commensal Streptococcus gordonii proteolytically cleave the salivary PRP-1 polypeptide into an RGRPQ innate peptide. The Arg and Gln termini are crucial for RGRPQ-mediated ammonia production and proliferation by S. gordonii SK12 and adhesion inhibition and desorption by Actinomyces naeslundii T14V, respectively. Here we have applied (i) a multivariate approach using RGRPQ-related peptides varied at amino acids 2, 3, and 4 simultaneously and (ii) size and N- and C-terminal modifications of RGRPQ to generate structure activity information. While the N-terminal arginine motif mediated ammonia production independent of peptide size, other responses required more or less full-length peptide motifs. The motifs for adhesion inhibition and desorption were the same. The adhesion and proliferation motifs required similarly a hydrophobic/low polarity amino acid 4 but differentially a hydrophilic or hydrophobic character of amino acids 2/3, respectively; polar peptides with small/hydrophilic and hydrophilic amino acids 2 and 3, respectively, had high adhesion inhibition/desorption activity, and lipophilic peptides with large/hydrophobic amino acids 2 and 3 had high proliferation activity. Accordingly, while RIWWQ had increased proliferation but abolished adhesion/desorption activity, peptides designed with hydrophilic amino acids 2 and 3 were predicted to behave in the opposite way. Moreover, a RGRPQ mimetic for all three responses should mimic small hydrophilic, large nitrogen-containing, and hydrophobic/low polarity amino acids 2, 3, and 4, respectively. Peptides fulfilling these criteria were 1-1.6-fold improved in all three responses. Thus, both mimetics and peptides with differential proliferation and adhesion activities may be generated for evaluation in biofilm models.     

Nutrient uptake by Candida albicans: the influence of cell surface mannoproteins.

Numerous ultrastructural and biochemical analyses have been performed to characterize the cell wall composition and structure of Candida albicans. However, little investigation has focused on how subtle differences in cell wall structure influence the intracellular transport of amino acids and monosaccharides. In this study C. albicans 4918 and ATCC 10231 were grown in culture conditions capable of modifying surface mannoproteins and induced surface hydrophobic or hydrophilic yeast cell wall states. Subcultures of these hydrophobic and hydrophilic yeasts were subsequently incubated with one of seven L-[3H] amino acids: glycine, leucine, proline, serine, aspartic acid, lysine, or arginine. The transport of [3H] mannose and [3H] N-acetyl-D-glucosamine were also investigated. This study revealed significant strain differences (P < or = 0.05) between hydrophilic and hydrophobic yeast transport of these nutrients throughout a 2 h incubation. Hydrophilic cultures of 4918 and ATCC 10231 transported nearly two times more (pmol mg-1 dry weight) proline, mannose, and N-acetyl-D-glucosamine than hydrophobic yeast. Hydrophobic cultures preferentially incorporated serine and aspartic acid in both these strains. Strain variation was indicated with the transport of leucine, lysine, and arginine, as follows: experiments showed that hydrophilic 4918 cultures selectively transported leucine, lysine, and arginine, whereas, the hydrophobic ATCC 10231 cultures incorporated these amino acids.     

Sequence analysis of the wall-associated protein precursor of Streptococcus mutans antigen A

The nucleotide sequence has been determined for the Streptococcus mutans wall-associated protein A (wapA) gene from serotype c strains Ingbritt and GS5. The nucleotide sequence for each wapA gene was virtually identical, although the gene from strain GS5 contained a 24 base pair deletion. A 29 amino acid signal peptide was specified by each wapA gene with a mature protein of 424 amino acids (Mr, 45,276) for strain Ingbritt and 416 amino acids (Mr, 44,846) for strain GS5. In the C-terminal region of the wall-associated protein A, considerable sequence similarity was found with the membrane anchor region of proteins from other Gram-positive organisms such as the group A streptococcal M protein and the group G streptococcal IgG binding protein. Adjacent to the proposed membrane anchor is a highly hydrophilic region which may span the cell wall; both sequence data and experimental evidence indicate the existence of a region immediately outside the wall at which proteolytic cleavage occurs to release antigen A of Mr 29,000 into the culture supernatant. Thus, the wall-associated protein A is a precursor of the 29,000 Mr antigen A.     

Enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid substitutions in the surface unit envelope glycoprotein.

Serial passage of the prototype (PR) cell-adapted Wyoming strain of equine infectious anemia virus (EIAV) in fetal donkey dermal (FDD) rather than fetal horse (designated fetal equine kidney [FEK]) cell cultures resulted in the generation of a variant virus strain which produced accelerated cytopathic effects in FDD cells and was 100- to 1,000-fold more sensitive to neutralizing antibodies than its parent. This neutralization-sensitive variant was designated the FDD strain. Although there were differences in glycosylation between the PR and FDD strains, passage of the FDD virus in FEK cells did not reduce its sensitivity to neutralizing antibody. Nucleotide sequencing of the region encoding the surface unit (SU) protein from the FDD strain revealed nine amino acid substitutions compared with the PR strain. Two of these substitutions resulted in changes in the polarity of charge, four caused the introduction of a charged residue, and three had no net change in charge. Nucleotide sequence analysis was extended to the region of the FDD virus genome encoding the extracellular domain of the transmembrane envelope glycoprotein (TM). Unlike the situation with the FDD virus coding region, there were minor variations in nucleotide sequence between individual molecular clones containing this region of the TM gene. Although each clone contained three nucleotide substitutions compared with the PR strain, only one of these was common to all, and this did not affect the amino acid content. Of the remaining two nucleotide substitutions, only one resulted in an amino acid change, and in each case, this change appeared to be conservative. To determine if amino acid substitutions in the SU protein of FDD cell-grown viruses were responsible for the enhanced sensitivity to neutralizing antibodies, chimeric viruses were constructed by using an infectious molecular clone of EIAV. These chimeric viruses contained all of the amino acid substitutions found in the FDD virus strain and were significantly more sensitive to neutralizing antibodies than viruses from the parental (PR) molecular clone. These results demonstrated that sensitivity to neutralizing antibodies in EIAV can be conferred by amino acid residues in the SU protein. However, such amino acid substitutions were not sufficient to enhance cytopathogenicity, as the chimeric viruses did not cause excessive degenererative effects in FDD cells, as was observed with the parental FDD virus strain.     

Complementary hydropathy and the evolution of interacting polypeptides

Summary It has been shown that codons coding for strongly hydrophilic amino acids are complemented by codons that code for strongly hydrophobic ones, leading to a hypothesis stating that peptides thus encoded should interact. Though the principle has been validated in a number of experimental models, its general applicability has been questioned. I have discussed this principle, showing that the correlation between coding and noncoding strand amino acids was maintained, indeed slightly improved, when weighted averages based on codon usage tables were used to determine noncoding strand amino acid hydropathies. The coding capacity of the noncoding strand and its content of open reading frames were also discussed. Another point of contention that was afforded further clarification is the chemical plausibility of interactions between hydrophobic and hydrophilic amino acids implicit in this concept. The extension of complementary domains was also dealt with. Finally, I have discussed what I called the evolutionary drift of primary structure, and I showed as an example that though nucleotide sequences coding for the substance K receptor bear little resemblance to the inverse complement of that which codes for the SK peptide, a peptide spanning residues 130–139 is hydropathically very similar to that predicted from such an inverse complement.     

Role of mutational bias and natural selection on genome-wide nucleotide bias in prokaryotic organisms

Correlations between genomic GC contents and amino acid frequencies were studied in the homologous sequences of 12 eubacterial genomes. Results show that amino acids encoded by GC-rich codons increases significantly with genomic GC contents, whereas opposite trend was observed in case of amino acids encoded by GC-poor codons. Further studies show all the amino acids do not change in the predicted direction according to their genomic GC pressure, suggesting that protein evolution is not entirely dictated by their nucleotide frequencies. Amino acid substitution matrix calculated among hydrophobic, amphipathic and hydrophilic amino acid groups' shows that amphipathic and hydrophilic amino acids are more frequently substituted by hydrophobic amino acids than from hydrophobic to hydrophilic or amphipathic amino acids. This indicates that nucleotide bias induces a directional changes in proteome composition in such a way that underwent strong changes in hydropathy values. In fact, significant increases in hydrophobicity values have also been observed with the increase of genomic GC contents. Correlations between GC contents and amino acid compositions in three different predicted protein secondary structures show that hydropathy values increases significantly with GC contents in aperiodic and helix structures whereas strand structure remains insensitive with the genomic GC levels. The relative importance of mutation and selection on the evolution of proteins have been discussed on the basis of these results.     

The complete amino-acid sequence of the large bacteriochlorophyll-binding polypeptide from light-harvesting complex II (B800-850) of Rhodopseudomonas capsulata

The large bacteriochlorophyll-a-binding polypeptide of the light-harvesting complex II (B800-850), having an apparent Mr with sodium dodecyl sulfate/polyacrylamide electrophoresis of 10000, has been isolated and purified from intracytoplasmic membranes of the phototrophically negative mutant strain Y5 of Rhodopseudomonas capsulata. The primary structure of this polypeptide has been determined. The polypeptide consists of 60 amino acid residues yielding an Mr of 7322. The hydrophobic stretch in positions 16-35 with a histidine in position 31 might be of importance for interaction with bacteriochlorophyll. The C-terminal part is also hydrophobic while the N-terminal part consists of hydrophilic amino acids. The polarity of the total amino acids was determined to be 28.3%.     

Sequential hydrophobic partitioning of cells of Pseudomonas aeruginosa gives rise to variants of increasing cell-surface hydrophobicity

The partitioning of bacterial cells in a dual aqueous-solvent phase system leads to separation into 'hydrophilic' and hydrophobic functions. Sequential multistep partitioning, accompanied by successive enrichment, gives rise to several cycles of hydrophobic and hydrophilic cell populations which possess different cell-surface hydrophobicity characteristics. Characterization of the cell-surface hydrophobicity by several methods (salting-out aggregation test, bacterial adherence to hydrocarbon, polystyrene binding and hydrophobic interaction chromatography) was carried out. The cell-surface hydrophobicity varied in the order: hydrophilic fraction < parental strain < first cycle hydrophobic variant < second cycle hydrophobic variant < third cycle hydrophobic variant. Electron microscopy showed that the most hydrophobic variant was densely covered by hydrophobic structures - fimbriae - whereas the parental strain was covered by a mixture of surface structures. The hydrophilic variant was covered by an amorphous exopolymeric substance, which is a polysaccharide, shown by its reaction with Alcian blue.     

Effect of hydrophobicity of utilization of peptides by ruminal bacteria in vitro

When mixed ruminal bacteria were incubated with a pancreatic casein hydrolysate and free amino acids of a similar composition, rates of ammonia production were much greater for peptides than for amino acids. The pancreatic digest of casein was then fractionated with 90% isopropyl alcohol. Hydrophobic peptides which dissolved in alcohol contained an abundance of phenolic and aliphatic amino acids, while the hydrophilic peptides which were precipitated by alcohol contained a large proportion of the highly charged amino acids. The Km values of the mixed ruminal bacteria for each fraction were similar (0.88 versus 0.98 g/liter), but the Vmax of the hydrophilic peptides was more than twice that of the hydrophobic peptides (18 versus 39 mg of NH3 per g of bacterial protein per h). Pure cultures of ruminal bacteria had a similar preference for hydrophilic peptides and likewise utilized peptides at a faster rate than free amino acids. Since peptide degradation rates differed greatly, hydrophobicity is likely to influence the composition of amino acids passing unfermented to the lower gut of ruminant animals.     

Effect of hydrophobicity of utilization of peptides by ruminal bacteria in vitro.

When mixed ruminal bacteria were incubated with a pancreatic casein hydrolysate and free amino acids of a similar composition, rates of ammonia production were much greater for peptides than for amino acids. The pancreatic digest of casein was then fractionated with 90% isopropyl alcohol. Hydrophobic peptides which dissolved in alcohol contained an abundance of phenolic and aliphatic amino acids, while the hydrophilic peptides which were precipitated by alcohol contained a large proportion of the highly charged amino acids. The Km values of the mixed ruminal bacteria for each fraction were similar (0.88 versus 0.98 g/liter), but the Vmax of the hydrophilic peptides was more than twice that of the hydrophobic peptides (18 versus 39 mg of NH3 per g of bacterial protein per h). Pure cultures of ruminal bacteria had a similar preference for hydrophilic peptides and likewise utilized peptides at a faster rate than free amino acids. Since peptide degradation rates differed greatly, hydrophobicity is likely to influence the composition of amino acids passing unfermented to the lower gut of ruminant animals.     

TRYPTIC PEPTIDES FROM BOVINE WHITE MATTER PROTEOLIPIDS

Abstract— The amino acid composition of the fractions obtained after tryptic digestion of performic acid oxidized and non-oxidized white matter proteolipids was studied. The acid-soluble fraction from the tryptic digest represented between 25 and 30% of the starting material and was relatively enriched in hydrophilic amino acids and deficient in hydrophobic amino acids. The acid-soluble peptides were separated by high voltage paper electrophoresis, and the amino acid compositions of 16 peptides were determined; three additional peptides were obtained from the acid-soluble digest of the oxidized proteolipid. The sequence of 7 peptides including the N- and C-terminal peptides is reported. The results suggest that the protein is segregated into hydrophilic and hydrophobic regions and that small hydrophilic regions are separated by large hydrophobic areas.     

Hydropathic anti-complementarity of amino acids based on the genetic code

An interesting pattern in the genetic code has been discovered. Codons for hydrophilic and hydrophobic amino acids on one strand of DNA are complemented by codons for hydrophobic and hydrophilic amino acids on the other DNA strand, respectively. The average tendency of codons for "uncharged" (slightly hydrophilic) amino acids is to be complemented by codons for "uncharged" amino acids.     

大黄鱼CD53基因克隆和分子特征分析

白细胞表面抗原CD53属于四跨膜蛋白超家族,在免疫反应中起着重要作用。在构建大黄鱼（Larim ichthyscrocea）肌肉组织cDNA文库的基础上,克隆了CD53基因。克隆到的CD53基因全长1210bp,其中5-′UTR 113bp,3′-UTR 422bp,CDS 675bp,编码224个氨基酸。生物信息学分析显示大黄鱼CD53存在4次跨膜结构,N端和C端都位于细胞膜内,膜外有两个亲水环,跨膜区域为疏水区域,两个N-糖基化位点都位于靠近C端的亲水环上。大黄鱼CD53氨基酸序列具非常高的保守性,与三刺鱼、斑马鱼、虹鳟等多种鱼类的相似性在70%以上。在检测的10种组织中,CD53只在大黄鱼的脾、骨骼肌、肾、肝、肠组织中表达,其中在肠组织中表达最强。     

Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity.

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.     

Debittering effect of Monascus carboxypeptidase during the hydrolysis of soybean protein

The actions of pepsin and the admixture of pepsin and Monascus pilosus carboxypeptidase 1 (MpiCP-1) on the hydrolysis of soybean protein were studied. The results showed that the pepsin hydrolyzate of soybean protein was much more bitter and contained relatively smaller amounts of total free amino acids than the hydrolyzate obtained with the admixture of pepsin and MpiCP-1. In addition, hydrophilic and hydrophobic amino acids were present in almost equal proportions in the pepsin hydrolyzate, while mainly hydrophobic amino acids made up the hydrolyzate obtained with the admixture of pepsin and MpiCP-1. These results suggest that MpiCP-1 suppresses and reverses the development of the bitterness taste that results from the pepsin hydrolysis of soybean protein by releasing mainly hydrophobic amino acids from the C-termini of the bitter components.     

Sequence analysis of the 17-kilodalton-antigen gene from Rickettsia rickettsii.

DNA obtained from the Sheila Smith strain of Rickettsia rickettsii was digested to completion with the restriction endonucleases BamHI and SalI and ligated with the plasmid vector pUC19. The ligation mixture was used to transform Escherichia coli. A total of 465 bacterial clones were screened for antigen production with hyperimmune rabbit serum. One of the reactive clones, containing a recombinant plasmid designated pSS124, was solubilized and subjected to immunoblot analysis and revealed expression of a 17-kilodalton protein reactive with anti-R. rickettsii serum that comigrated with an antigen from R. rickettsii. A 1.6-kilobase PstI-BamHI fragment from pSS124 was subcloned and continued to direct synthesis of the 17-kilodalton antigen. The nucleotide sequence was determined for this 1.6-kilobase subclone, which encompassed the gene encoding the polypeptide as well as flanking regions containing potential regulatory sequences. The open reading frame consisted of 477 nucleotides that specified a 159-amino-acid protein with a calculated molecular weight of 16,840. The deduced amino acid sequence contained a hydrophobic sequence near the amino terminus that resembled signal peptides described for E. coli. The carboxy terminus was hydrophilic in nature and probably contained the exposed epitopes.     

Molecular Evolution of Amelogenin in Mammals

An evolutionary analysis of mammalian amelogenin, the major protein of forming enamel, was conducted by comparison of 26 sequences (including 14 new ones) representative of the main mammalian lineages. Amelogenin shows highly conserved residues in the hydrophilic N- and C-terminal regions. The central hydrophobic region (most of exon 6) is more variable, but it has conserved a high amount of proline and glutamine located in triplets, PXQ, indicating that these residues play an important role. This region evolves more rapidly, and is less constrained, than the other well-conserved regions, which are subjected to strong constraints. The comparison of the substitution rates in relation to the CpG richness confirmed that the highly conserved regions are subjected to strong selective pressures. The amino acids located at important sites and the residues known to lead to amelogenesis imperfecta when substituted were present in all sequences examined. Evolutionary analysis of the variable region of exon 6 points to a particular zone, rich in either amino acid insertion or deletion. We consider this region a hot spot of mutation for the mammalian amelogenin. In this region, numerous triplet repeats (PXQ) have been inserted recently and independently in five lineages, while most of the hydrophobic exon 6 region probably had its origin in several rounds of triplet insertions, early in vertebrate evolution. The putative ancestral DNA sequence of the mammalian amelogenin was calculated using a maximum likelihood approach. The putative ancestral protein was composed of 177 residues. It already contained all important amino acid positions known to date, its hydrophobic variable region was rich in proline and glutamine, and it contained triplet repeats PXQ as in the modern sequences.Reviewing Editor: Dr. Cecilia Saccone