Structural plasticity of the phage P22 tail needle gp26 probed with xenon gas

The tail needle, gp26, is a highly stable homo‐trimeric fiber found in the tail apparatus of bacteriophage P22. In the mature virion, gp26 is responsible for plugging the DNA exit channel, and likely plays an important role in penetrating the host cell envelope. In this article, we have determined the 1.98 Å resolution crystal structure of gp26 bound to xenon gas. The structure led us to identify a calcium and a chloride ion intimately bound at the interior of α‐helical core, as well as seven small cavities occupied by xenon atoms. The two ions engage in buried polar interactions with gp26 side chains that provide specificity and register to gp26 helical core, thus enhancing its stability. Conversely, the distribution of xenon accessible cavities correlates well with the flexibility of the fiber observed in solution and in the crystal structure. We suggest that small internal cavities in gp26 between the helical core and the C‐terminal tip allow for flexible swinging of the latter, without affecting the overall stability of the protein. The C‐terminal tip may be important in scanning the bacterial surface in search of a cell‐envelope penetration site, or for recognition of a yet unidentified receptor on the surface of the host.     

Primary structure of the tail sheath protein of bacteriophage T4 and its gene

Complete sequence determination of gene 18 encoding the tail sheath protein was carried out mainly by the Maxam-Gilbert method. Approximately 40 peptides contained in a tryptic digest and a lysyl endopeptidase digest of gp 18 were isolated by reversed-phase high-performance liquid chromatography. All the peptides were identified along the nucleotide sequence of gene 18 based on the amino acid compositions. These peptides cover 88% of the total primary structure. Furthermore, the amino acid sequences of 9 of the 40 peptides were determined by a gas-phase protein sequencer; one of them turned to be the N-terminal one. The C-terminal peptide in the tryptic digest was isolated from the unadsorbed fraction of affinity chromatography on immobilized anhydrotrypsin and the amino acid sequence was also determined. Thus, the complete primary structure of gp 18 was determined; it has 658 amino acid residues and a molecular weight of 71,160.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Engineering of bacteriophage T4 tail sheath protein

Gene product 18 (gp18, 659 amino acids) forms bacteriophage T4 contractile tail sheath. Recombinant protein assembles into different length polysheaths during expression in the cell, which complicates the preparation of protein crystals for its spatial structure determination. To design soluble monomeric gp18 mutants unable to form polysheaths and useful for crystallization, we have used Bal31 nuclease for generation deletions inside gene 18 encoding the Ile507-Gly530 region. Small deletions in the region of Ile507-Ile522 do not affect the protein assembly into polysheaths. Protein synthesis termination occurs because of reading frame failure in the location of deletions. Some fragments of gp18 containing short pseudoaccidental sequence in the C-terminal, while being soluble, have lost the ability for polysheath assembly. For the first time we succeeded in obtaining crystals of a soluble gp18 fragment containing 510 amino acids which, according to trypsin resistance, is similar to native protein monomer.     

Chaperonin-mediated folding of bacteriophage T4 major capsid protein. II. Production of gene product 23 deletion mutants

Folding of bacteriophage T4 major capsid protein, gene product 23 (534 a.a.), is aided by two proteins: E. coli GroEL chaperonin and viral gp31 co-chaperonin. In the present work a set of mutants with extensive deletions inside gene 23 using controlled digestion with Bal31 nuclease has been constructed. Proteins with deletions were co-expressed from plasmid vectors with phage gp31 co-chaperonin. Deletions from 8 to 33 a.a. in the N-terminal region of the gp23 molecule covering the protein proteolytic cleavage site during capsid maturation have no influence on the mutants' ability to produce in E. coli cells proteins which form regular structures—polyheads. Deletions in other regions of the polypeptide chain (187-203 and 367-476 a.a.) disturb the correct folding and subsequent assembly of gp23 into polyheads.     

Control of bacteriophage T4 tail lysozyme activity during the infection process

Bacteriophage T4 has an efficient mechanism for injecting the host Escherichiacoli cell with genomic DNA. Its gene product 5 (gp5) has a needle-like structure attached to the end of a tube through which the DNA passes on its way out of the head and into the host. The gp5 needle punctures the outer cell membrane and then digests the peptidoglycan cell wall in the periplasmic space. gp5 is normally post-translationally cleaved between residues 351 and 352. The function of this process in controlling the lysozyme activity of gp5 has now been investigated. When gp5 is over-expressed in E.coli, two mutants (S351H and S351A) showed a reduction of cleavage products and five other mutants (S351L, S351K, S351Y, S351Q, and S351T) showed no cleavage. Furthermore, in a complementation assay at 20 degrees C, the mutants that had no cleavage of gp5 produced a reduced number of plaques compared to wild-type T4. The crystal structure of the non-cleavage phenotype mutant of gp5, S351L, complexed with gene product 27, showed that the 18 residues in the vicinity of the potential cleavage site (disordered in the wild-type structure) had visible electron density. The polypeptide around the potential cleavage site is exposed, thus allowing access for an E.coli protease. The lysozyme activity is inhibited in the wild-type structure by a loop from the adjacent gp5 monomer that binds into the substrate-binding site. The same inhibition is apparent in the mutant structure, showing that the lysozyme is inhibited before gp5 is cleaved and, presumably, the lysozyme is activated only after gp5 has penetrated the outer membrane.     

Differential expression of influenza N protein and neuraminidase antigenic determinants in Escherichia coli

Two influenza gene products of similar size and codon usage have been expressed in Escherichia coli under control of the phage lambda pR promoter. The influenza N protein (NP) was expressed in its entirety after fusion to a short (12 amino acid) segment of the lambda cro gene product and constituted about 1-2% of total soluble cell protein after induction. By contrast, constructions using the full length neuraminidase (NA) gene failed to give rise to detectable amounts of NA antigen after fusion to either the 12 amino acid Cro peptide or after fusion to bacterial beta-galactosidase (beta gal). Rather, expression of NA antigenic determinants was only achieved after deletion of coding sequences at the 3' end of the beta gal-NA fusion construct such that the encoded protein precipitated within the cell.     

Overexpression of klotho protein modulates uninephrectomy-induced compensatory renal hypertrophy by suppressing IGF-I signals

The klotho gene is highly expressed in the distal convoluted tubule of the kidney, while its encoded protein has many physiological and pathophysiological renal roles. We investigated the effect of klotho protein on physiological compensatory renal hypertrophy after nephrectomy in klotho transgenic (KLTG) mice. Renal hypertrophy was suppressed in KLTG mice compared with wild-type mice, and this was associated with suppression of insulin growth factor-1 (IGF-1) signaling by klotho protein. In vitro, IGF-1 signaling was suppressed in human proximal tubular cells transfected with the klotho plasmid. Our data suggest that klotho modulates compensatory renal hypertrophy after nephrectomy via suppression of the IGF-1 signaling pathway, indicating a novel physiological role for klotho protein in the kidney.     

Insertion of a chaperone domain converts FKBP12 into a powerful catalyst of protein folding

The catalytic activity of human FKBP12 as a prolyl isomerase is high towards short peptides, but very low in proline-limited protein folding reactions. In contrast, the SlyD proteins, which are members of the FKBP family, are highly active as folding enzymes. They contain an extra "insert-in-flap" or IF domain near the prolyl isomerase active site. The excision of this domain did not affect the prolyl isomerase activity of SlyD from Escherichia coli towards short peptide substrates but abolished its catalytic activity in proline-limited protein folding reactions. The reciprocal insertion of the IF domain of SlyD into human FKBP12 increased its folding activity 200-fold and generated a folding catalyst that is more active than SlyD itself. The IF domain binds to refolding protein chains and thus functions as a chaperone module. A prolyl isomerase catalytic site and a separate chaperone site with an adapted affinity for refolding protein chains are the key elements for a productive coupling between the catalysis of prolyl isomerization and conformational folding in the enzymatic mechanisms of SlyD and other prolyl isomerases, such as trigger factor and FkpA.     

Expression,stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.     

Soluble components of the flagellar export apparatus,FliI, FliJ,and FliH,do not deliver flagellin,the major filament protein,from the cytosol to the export gate

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook–filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

Plasticity of the gene functions for DNA replication in the T4-like phages

We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter johnsonii phage 133 and Vibrio natriegens phage nt-1. Each of these phage genomes exhibited a unique sequence that distinguished it from its relatives, although there were examples of genomes that are very similar to each other. As a group the phages compared here diverge from one another by several criteria, including (a) host range, (b) genome size in the range between approximately 160 kb and approximately 250 kb, (c) content and genetic organization of their T4-like genes for DNA metabolism, (d) mutational drift of the predicted T4-like gene products and their regulatory sites and (e) content of open-reading frames that have no counterparts in T4 or other known organisms (novel ORFs). We have observed a number of DNA rearrangements of the T4 genome type, some exhibiting proximity to putative homing endonuclease genes. Also, we cite and discuss examples of sequence divergence in the predicted sites for protein-protein and protein-nucleic acid interactions of homologues of the T4 DNA replication proteins, with emphasis on the diversity in sequence, molecular form and regulation of the phage-encoded DNA polymerase, gp43. Five of the sequenced phage genomes are predicted to encode split forms of this polymerase. Our studies suggest that the modular construction and plasticity of the T4 genome type and several of its replication proteins may offer resilience to mutation, including DNA rearrangements, and facilitate the adaptation of T4-like phages to different bacterial hosts in nature.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).     

Polypeptide composition of a Photosystem II core complex. Presence of a herbicide-binding protein

The polypeptide composition of a Photosystem II (PS II) core complex from higher plant chloroplasts has been characterized by subjecting the isolated complex to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides in the 40–50 kDa size class, attributed to the chlorophyll a-binding apoproteins of PS II, were resolved when the urea concentration in the SDS-polyacrylamide gel electrophoresis was greater than 1 M. The two chlorophyll a-binding proteins were dissimilar in their primary structure based upon their different hydrolysis products on SDS-polyacrylamide gel electrophoresis following papain treatment. The core complex contained three additional polypeptides. Two polypeptides in the 30–34 kDa size class were resolved when the urea concentration in the gel system was increased to greater than 4 M. One of the polypeptides in this size class was identified as the herbicide-binding protein from azido[¹⁴C]atrazine labeling studies. The herbicide-binding protein displayed an anomalous electrophoretic migration behavior in SDS-polyacrylamide gel electrophoresis in the presence or absence of urea; its apparent molecular weight decreased when the urea concentration increased. The fifth protein component of the core complex was attributed to cytochrome b-559 which was found to consist of the ascorbate- and dithionite-reducible forms in the samples prior to SDS solubilization.     

Calmodulin as a protein linker and a regulator of adaptor/scaffold proteins

Calmodulin (CaM) is a universal regulator for a huge number of proteins in all eukaryotic cells. Best known is its function as a calcium-dependent modulator of the activity of enzymes, such as protein kinases and phosphatases, as well as other signaling proteins including membrane receptors, channels and structural proteins. However, less well known is the fact that CaM can also function as a Ca^2 +-dependent adaptor protein, either by bridging between different domains of the same protein or by linking two identical or different target proteins together. These activities are possible due to the fact that CaM contains two independently-folded Ca^2 + binding lobes that are able to interact differentially and to some degree separately with targets proteins. In addition, CaM can interact with and regulates several proteins that function exclusively as adaptors. This review provides an overview over our present knowledge concerning the structural and functional aspects of the role of CaM as an adaptor protein and as a regulator of known adaptor/scaffold proteins.     

Specificity of the initial collapse in the folding of the cold shock protein

The two-state folding reaction of the cold shock protein from Bacillus caldolyticus (Bc-Csp) is preceded by a rapid chain collapse. A fast shortening of intra-protein distances was revealed by F?rster resonance energy transfer (FRET) measurements with protein variants that carried individual pairs of donor and acceptor chromophores at various positions along the polypeptide chain. Here we investigated the specificity of this rapid compaction. Energy transfer experiments that probed the stretching of strand beta2 and the close approach between the strands beta1 and beta2 revealed that the beta1-beta2 hairpin is barely formed in the collapsed form, although it is native-like in the folding transition state of Bc-Csp. The time course of the collapse could not be resolved by pressure or temperature jump experiments, indicating that the collapsed and extended forms are not separated by an energy barrier. The co-solute (NH4)2SO4 stabilizes both native Bc-Csp and the collapsed form, which suggests that the large hydrated SO4(2-) ions are excluded from the surface of the collapsed form in a similar fashion as they are excluded from folded Bc-Csp. Ethylene glycol increases the stability of proteins because it is excluded preferentially from the backbone, which is accessible in the unfolded state. The collapsed form of Bc-Csp resembles the unfolded form in its interaction with ethylene glycol, suggesting that in the collapsed form the backbone is still accessible to water and small molecules. Our results thus rule out that the collapsed form is a folding intermediate with native-like chain topology. It is better described as a mixture of compact conformations that belong to the unfolded state ensemble. However, some of its structural elements are reminiscent of the native protein.     

A rapid method for the isolation and characterization of a homogeneous population of sterptococcal Fc receptors

Immunoblotting techniques were developed and used to determine the most suitable conditions for extracting bacterial receptors for the Fc region of human IgG. Crude extracts of a group C streptococcus were separated on 10% polyacrylamide SDS slab gels, electroblotted onto a nitrocellulose membrane, probed with radioiodinated human IgG containing unlabeled F(ab)₂ fragments and visualized by autoradiography. This procedure enabled us to compare size, heterogeneity and quantity of functionally active Fc receptors in crude extracts. Although more total Fc receptor could be extracted by phage lysis or mutanolysin treatment, only treatment of the group C streptococcus with trypsin, under suboptimal pH conditions for enzyme activity, resulted in a homogeneous product. The yield of affinity purified Fc receptor was 64 μg/g (wet weight) of bacteria. The affinity purified protein had a molecular weight of 40 000 and retained its ability to bond to the Fc region of IgG. The methods described are also applicable to the isolation of Fc receptors from other bacterial sources.     

Optimization of the gbeta1 domain by computational design and by in vitro evolution: structural and energetic basis of stabilization

Computational design and in vitro evolution are major strategies for stabilizing proteins. For the four critical positions 16, 18, 25, and 29 of the B domain of the streptococcal protein G (Gbeta1), they identified the same optimal residues at positions 16 and 25, but not at 18 and 29. Here we analyzed the energetic contributions of the residues from these two approaches by single and double mutant analyses and determined crystal structures for a variant from the calculation (I16/L18/E25/K29) and from the selection (I16/I18/E25/F29). The structural analysis explains the observed differences in stabilization. Residues 16, 18, and 29 line an invagination, which results from a packing defect between the helix and the beta-sheet of Gbeta1. In all stabilized variants, residues with larger side-chains occur at these positions and packing is improved. In the selected variant, packing is better optimized than in the computed variant. Such differences in side-chain packing strongly affect stability but are difficult to evaluate by computation.     

Association of SNP41, SNP56 and a novel SNP in PDE4D gene with stroke and its subtypes

An association between phosphodiesterase 4D (PDE4D) gene and risk of stroke has been suggested by deCODE group in an Icelandic population. In the present case–control study we investigated the association of SNP41 (rs12153798) and SNP56 (rs702553) with ischemic stroke and stroke subtypes. Five hundred and sixteen ischemic stroke patients and 513 healthy age and sex matched controls were included in the study. The genotypes were determined by subjecting the PCR products to sequencing. Both the SNPs 56 and 41 associated significantly with stroke [adjusted OR = 1.97; 95% CI (1.262–3.082); p = 0.003: adjusted OR = 5.42; 95% CI (3.45–8.5); p < 0.001 respectively]. In addition to this, a novel SNP at position 59736747 T > G was found while sequencing the PCR products including SNP56. This novel SNP was found in patients as well as controls but did not show a significant association with the disease. We found significant association of SNPs 56 and 41 with large artery atherosclerosis, lacunar and cardioembolic stroke. In conclusion PDE4D gene plays a key part in the pathogenesis of ischemic stroke in the South Indian population from Andhra Pradesh.