The formation of a native-like structure containing eight conserved hydrophobic residues is rate limiting in two-state protein folding of ACBP.

The acyl-coenzyme A-binding proteins (ACBPs) contain 26 highly conserved sequence positions. The majority of these have been mutated in the bovine protein, and their influence on the rate of two-state folding and unfolding has been measured. The results identify eight sequence positions, out of 24 probed, that are critical for fast productive folding. The residues are all hydrophobic and located in the interface between the N- and C-terminal helices. The results suggest that one specific site dominated by conserved hydrophobic residues forms the structure of the productive rate-determining folding step and that a sequential framework model can describe the protein folding reaction.     

The primary structure of a 4.0-kDa photosystem I polypeptide encoded by the chloroplast psaI gene

Partial amino acid sequences have been determined for a 4.0-kDa photosystem I polypeptide from barley. A comparison with the sequence of the chloroplast genome of Nicotiana tabacum and Marchantia polymorpha identified the polypeptide as chloroplast-encoded. We designate the corresponding gene psaI and the polypeptide PSI-I. The barley chloroplast psaI gene was sequenced. The gene encodes a polypeptide of 36 amino acid residues with a deduced molecular mass of 4008 Da. The 4.0-kDa polypeptide is N-terminally blocked with a formyl-methionine residue. Plasma desorption mass spectrometry established that the polypeptide is not post-translationally processed except for possible conversion of a methionine residue into methionine sulfone. The hydrophobic 4.0-kDa polypeptide is predicted to have one membrane-spanning alpha-helix and is homologous to transmembrane helix E of the D2 reaction center polypeptide of photosystem II.     

The primary structure of rat ribosomal protein L8.

The amino acid sequence of the rat 60S ribosomal subunit protein L8 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L8 has 257 amino acids and has a molecular weight of 28,007. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 4 or 5 copies of the L8 gene. The mRNA for the protein is about 950 nucleotides in length. Rat L8 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes.     

The primary structure of rat ribosomal protein S8.

The amino acid sequence of rat ribosomal protein S8 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2- and carboxyl-terminal amino acid sequences of the protein. Ribosomal protein S8 contains 207 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 23,928. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7-9 copies of the S8 gene. Ribosomal protein S8 contains a possible internal repeat that has 12 or 13 residues, is basic, and occurs 5 times in the protein.     

Correlation of virus polypeptide structure with attenuation of poliovirus type 1.

Several avirulent samples of poliovirus type 1 derived in the process attenuating the neurovirulent Mahoney strain show an altered virus capsid polypeptide, VP-1, on polyacrylamide gel electrophoresis of sodium dodecyl sulfate-disrupted virions.     

The isolation and primary structure of a 22-kDa extracellular matrix protein from bovine skin

The primary structure of a 22-kDa protein which was isolated during the purification of bovine skin dermatan sulfate proteoglycan is described. The uronate-rich fraction from DEAE-Sepharose chromatography of a 7.8 M urea extract of bovine fetal skin was subjected to gel filtration on Sepharose CL-6B in 4 M guanidine HCl. A prominent component of mass 22 kDa was separated from the proteoglycan and further purified on octyl-Sepharose. The primary structure of this component was determined and found to contain three repeat regions. Each of the three sections contains a similar pattern of looped disulfide bonds. A six-amino acid consensus sequence, Asp-Arg-Glx-Trp-Asn/Gln/Lys-Phe/Tyr, is found in each loop. This domain may be involved in associations of the molecule with other extracellular matrix components.     

The primary structure of the 32-kDa subunit of human replication protein A

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.     

The gene encoding the hydrophobic surfactant protein SP-C is located on 8p and identifies an EcoRI RFLP.

Pulmonary surfactant is composed primarily of phospholipids but also contains three known surfactant-specific proteins. These proteins are important in determining the physical properties of pulmonary surfactant--including its ability to adsorb to an air-liquid interface and its structure--but also appear to influence surfactant metabolism. We have previously assigned two surfactant proteins, SP-A (a 28-36-kDa glycoprotein) and SP-B (an 18-kDa hydrophobic protein), to the short arm of chromosome 10 and to chromosome 2, respectively. We now report that the gene encoding the 5-8 kDa hydrophobic surfactant protein SP-C is located on the short arm of chromosome 8. A cDNA clone encoding the entire protein recognizes a useful EcoRI restriction-site-length polymorphism. Evaluation of congenital syndromes manifesting autosomal abnormalities does not further elucidate the functional role of this protein in promoting normal respiratory physiology.     

Soybean hydrophobic protein. Isolation, partial characterization and the complete primary structure

A 9000-Mr protein isolated from a 60% ethanolic extract of soybean (Glycine max) seeds has been characterized and fully sequenced. The protein consists of 80 amino acid residues with four disulfide bonds. It contains a large number of hydrophobic residues and lacks methionine, phenylalanine, tryptophan, lysine and histidine residues. The protein readily crystallizes from water but is quite soluble in aqueous organic solvents like 95% 1-propanol. It aggregates to form large molecules (above 80 kDa) under ordinary denaturing conditions, such as 6 M guanidine X HCl and 8 M urea. Sequence analysis showed that the amino-terminal four-fifths is extremely hydrophobic and most of the acidic residues exist as their amide forms, and only the carboxyl-terminal short segment is rather hydrophilic. A computer search for homology detected an unexpected similarity of this protein to rat prolactin; however, its significance could not be assessed and this protein appears to represent a hitherto unknown protein family. Although no biochemical activity could be detected, the existence in relatively high abundance (approx. 200 mg from 1 kg seeds) of this novel protein may suggest its physiological significance in the plant.     

Contribution of hydrophobic residues to ice binding by fish type III antifreeze protein

Type III antifreeze protein (AFP) is a 7-kDa globular protein with a flat ice-binding face centered on Ala 16. Neighboring hydrophilic residues Gln 9, Asn 14, Thr 15, Thr 18 and Gln 44 have been implicated by site-directed mutagenesis in binding to ice. These residues have the potential to form hydrogen bonds with ice, but the tight packing of side chains on the ice-binding face limits the number and strength of possible hydrogen bond interactions. Recent work with alpha-helical AFPs has emphasized the hydrophobicity of their ice-binding sites and suggests that hydrophobic interactions are important for antifreeze activity. To investigate the contribution of hydrophobic interactions between type III AFP and ice, Leu, Ile and Val residues on the rim of the ice-binding face were changed to alanine. Mutant AFPs with single alanine substitutions, L19A, V20A, and V41A, showed a 20% loss in activity. Doubly substituted mutants, L19A/V41A and L10A/I13A, had less than 50% of the activity of the wild type. Thus, side chain substitutions that leave a cavity or undercut the contact surface are almost as deleterious to antifreeze activity as those that lengthen the side chain. These mutations emphasize the importance of maintaining a specific surface contour on the ice-binding face for docking to ice.     

The isolation of human-erythrocyte band-3 polypeptide labelled with a photosensitive hydrophobic probe.

To investigate the intramembranous domains of the major band-3 polypeptide, human erythrocyte membranes were labelled with 1-azido-4-[125I]iodobenzene. The anion-exchange protein has been isolated by a new procedure that decreases possible contamination by other integral membrane proteins of similar molecular weight. After chemical modification, the protein was subjected to proteolytic cleavage by several proteinases to yield a distinct pattern of peptides, a few of which contained radioactivity.     

Distribution of amino acid residues in the primary protein structure

The amino acid composition and sequence in primary structure of 180 proteins have been studied. It is shown that the distribution of amino acid residues is near to a random one, i.e. it is determined by the amino acid composition. The ratio between statistical and unique character of protein primary structures has been discussed. The amino acid sequence is suggested to be unique in fibrous proteins. In contrast the amino acid sequence in globular proteins is a statistical one. The statistical character of amino acids distribution in globular proteins explains the possibility of sensible text generation under the frame shift mutations, deletions and insertions.     

Identification of highly methylated arginine residues in an endogenous 20-kDa polypeptide in cancer cells.

Enzymatic methylation of endogenous proteins in several cancer cell lines was investigated to understand a possible relationship between protein-arginine methylation and cellular proliferation. Cytosolic extracts prepared from several cancer cells (HeLa, HCT-48, A549, and HepG2) and incubated with S-adenosyl-L-[methyl-3H]methionine revealed an intensely [methyl-3H]-labeled 20-kDa polypeptide. On the other hand, cytosolic extracts prepared from normal colon cells did not show any methylation of the 20-kDa protein under identical conditions. To identify nature of the 20-kDa polypeptide, purified histones were methylated with HCT-48 cytosolic extracts and analyzed by SDS-PAGE. However, none of the histones comigrated with the methylated 20-kDa polypeptide, indicating that it is unlikely to be any of the histone subclasses. The [methyl-3H]group in the 20-kDa polypeptide was stable at pH 10-11 (37 degrees C for 30 min) and methylation was not stimulated by GTPgammaS (4 mM), thus the reaction is neither carboxyl methylesterification on isoaspartyl residues, nor on C-terminal farnesylated cysteine. The present study together with the previous identification of N(G)-methylated arginine residues in the HCT-48 cytosol fraction suggests that this novel endogenous 20-kDa arginine-methylation is a cellular proliferation-related posttranslational modification reaction.     

The primary structure of a major polypeptide component from the venom of Naja melanoleuca.

The venom of the forest cobra, Naja melanoleuca, contains a number of homologous polypeptides containing between 60 and 71 amino acid resides. The primary structure of a major component (approx. 10% by weight of the crude venom) has been determined unambiguously. The molecule contians 61 amino acid residues and four disulphide bridges. It has not effect on neuromuscular transmission or the excitatory or inhibitory responses to acetylcholine of molluscan neurons. The molecule is similar to, but not identical with, the so-called cytotoxins VII2 and VII3 isolated, by others, from the same venom but reported to be minor components.     

Interfacial adsorption of simple lipid mixtures combined with hydrophobic surfactant protein from pig lung.

Hydrophobic pulmonary surfactant protein enriched in SP-C has been mixed in amounts up to 10% by weight with various phospholipids. The lipids used were dipalmitoyl phosphatidylcholine (DPPC), or DPPC plus unsaturated phosphatidylglycerol (PG), or phosphatidylinositol (PI) in molar ratios of 9:1 and 7:3. The protein enhanced the rate and extent of adsorption of each lipid preparation into the air-water interface, and its respreading after compression on a surface balance. Maximum surface pressures attained on compression of monolayers of mixtures of lipids were slightly higher in the presence of protein. The effects on rate and extent of adsorption were proportional to the amount of protein present. Mixtures containing 30 mol% PG or PI adsorbed more readily into the interface than those containing 10% acidic lipid or DPPC alone. Mixtures containing 30% PI were slightly more rapidly adsorbed than those containing 30% PG. The results suggest that mixtures of DPPC with either acidic lipid in the presence of surfactant protein could be effective in artificial surfactants.     

The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments.

Component 8c-1, one of four highly homologous component-8 subunit proteins present in the microfibrils of wool, was isolated as its S-carboxymethyl derivative and its amino acid sequence was determined. Large peptides were isolated after cleaving the protein chemically or enzymically and the sequence of each was determined with an automatic Sequenator. The peptides were ordered by sequence overlaps and, in some instances, by homology with known sequences from other component-8 subunits. The C-terminal residues were identified by three procedures. Full details of the various procedures used have been deposited as Supplementary Publication SUP 50133 (4 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. The result showed that the protein comprises 412 residues and has an Mr, including the N-terminal acetyl group, of 48,300. The sequence of residues 98-200 of component 8c-1 was found to correspond to the partial or complete sequences of four homologous type I helical segments previously isolated from helical fragments recovered from chymotryptic digests of microfibrillar proteins of wool [Crewther & Dowling (1971) Appl. Polym. Symp. 18, 1-20; Crewther, Gough, Inglis & McKern (1978) Text. Res. J. 48, 160-162; Gough, Inglis & Crewther (1978) Biochem. J. 173, 385]. Considered in relation to amino acid sequences of other intermediate-filament proteins, the sequence is in accord with the view that keratin filament proteins are of two types [Hanukoglu & Fuchs (1983) Cell (Cambridge, Mass.) 33, 915-924]. Filament proteins from non-keratinous tissues, such as desmin, vimentin, neurofilament proteins and the glial fibrillary acidic protein, which form monocomponent filaments, constitute a third type. It is suggested that as a whole the proteins from intermediate filaments be classed as filamentins, the three types at present identified forming subgroups of this class. The significant homologies between types I, II and III occur almost exclusively in segments of the chain that have been identified as having a coiled-coil structure together with the relatively short sections connecting these segments. The non-coiled-coil segments at the C- and N-termini show no significant homology between types, nor is homology in these segments apparent in all members of one type. Component 8c-1 does not show homology in its terminal segments with the known sequence of any other filamentin.(ABSTRACT TRUNCATED AT 400 WORDS)     

The optimal fraction of hydrophobic residues required to ensure protein collapse

The hydrophobic interaction is the main driving force for protein folding. Here, we address the question of what is the optimal fraction, f of hydrophobic (H) residues required to ensure protein collapse. For very small f (say f<0.1), the protein chain is expected to behave as a random coil, where the H residues are "wrapped" locally by polar (P) residues. However, for large enough f this local coverage cannot be achieved and the thermodynamic alternative to avoid contact with water is burying the H residues in the interior of a compact chain structure. The interior also contains P residues that are known to be clustered to optimize their electrostatic interactions. This means that the H residues are clustered as well, i.e. they effectively attract each other like the H-monomers in Dill's HP lattice model. Previously, we asked the question: assuming that the H monomers in the HP model are distributed randomly along the chain, what fraction of them is required to ensure a compact ground state? We claimed there that f approximately p(c), where p(c) is the site percolation threshold of the lattice (in a percolation experiment, each site of an initially empty lattice is visited and a particle is placed there with a probability p. The interest is in the critical (minimal) value, p(c), for which percolation occurs, i.e. a cluster connecting the opposite sides of the lattice is created). Due to the above correspondence between the HP model and real proteins (and assuming that the H residues are distributed at random) we suggest that the experimental f should lead to percolating clusters of H residues over the highly dense protein core, i.e. clusters of the core size. To check this theory, we treat a simplified model consisting of H and P residues represented by their alpha-carbon atoms only. The structure is defined by the C(alpha)-C(alpha) virtual bond lengths, angles and dihedral angles, and the X-ray structure is best-fitted onto a face-centered cubic lattice. Percolation experiments are carried out for 103 single-chain proteins using six different hydrophobic sets of residues. Indeed, on average, percolating clusters are generated, which supports our theory; however, some sets lead to a better core coverage than others. We also calculate the largest actual hydrophobic cluster of each protein and show that, on average, these clusters span the core, again in accord with our theory. We discuss the effect of protein size, deviations from the average picture, and implications of this study for defining reliable simplified models of proteins.     

Processing of surfactant protein C requires a type II transmembrane topology directed by juxtamembrane positively charged residues

Surfactant protein C (SP-C) is a lung-specific protein that is synthesized as a 21-kDa integral membrane propeptide (pro-SP-C) and proteolytically processed to a 3.7-kDa secretory product. Previous studies have shown that palmitoylation of pro-SP-C is dependent on two N-terminal juxtamembrane positively charged residues. We hypothesized that these residues influence modification of pro-SP-C by directing transmembrane orientation. Double substitution mutation of these juxtaposed residues from positive to neutral charged species resulted in complete reversal of transmembrane orientation of pro-SP-C and total abrogation of post-translational processing. Mutation of a single residue resulted in mixed orientation. Protein trafficking studies in A549 cells showed that while the double mutant was retained in the endoplasmic reticulum, single mutants produced a mixed pattern of both endoplasmic reticulum (double mutant-like) and vesicular (wild type-like) expression. Our study demonstrates the crucial role juxtamembrane positively charged residues play in establishing membrane topology and their influence on the trafficking and processing of pro-SP-C. Moreover this study provides a likely precedent for a mechanism in disorders associated with mutations in the membrane-flanking region of integral membrane proteins.     

The primary structure of a plant storage protein: zein.

The protein sequence of a representative of the zeins, the major storage proteins of maize, has been derived from the nucleotide sequence of a zein cDNA clone. This cDNA was sequence both by the Maxam and Gilbert and the M13-dideoxy techniques. The nucleotide sequence encompasses the non-translated 3' terminus of the mRNA, the entire coding sequence specifying both the mature zein protein and a small signal peptide, and a portion of the non-translated 5' region. The deduced amino acid composition and the amino-terminal amino acid sequence closely resemble those derived from chemical analysis of the zein protein fraction. The data presented represent the first complete amino acid sequence of a plant storage protein.