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PubMed2003

Overexpression, purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16.

The E5 proteins of human papillomavirus (HPV) are highly hydrophobic transmembrane proteins that display weak transforming activity. The HPV E5 proteins are localized largely to intracellular membranes, such as the Golgi apparatus and endoplasmic reticulum, but also appear in the plasma membrane. Infection with HPV16 is the cause of over 90% of human cervical cancers. HPV E5 is known to interact with growth factor receptors and gap junction proteins and is believed to play a role during the initiation of neoplasia. The structure of HPV E5 and the mechanism of its interactions with growth factor receptors remain largely unknown. In the present studies, the E5 protein of HPV16 was cloned into the pBAD/TOPO vector fused to an N-terminal thioredoxin leader and a C-terminal His-tag, and expressed in Escherichia coli. The identity of the protein was confirmed by immunoblotting using antibodies against a V5-epitope tag engineered into the protein. Due to formation of high molecular mass superaggregates of the protein, two chromatography steps were employed for its purification: (1) gel filtration chromatography to separate the superaggregated protein from other soluble proteins and (2) Ni-chelate affinity chromatography in the presence of detergent. The superaggregates of the E5-fusion protein were broken down to monomers and various oligomers by sonication in the presence of 0.2% SDS. The purified E5-fusion protein was then reconstituted into lipid vesicles and initial structural analysis of the protein was performed using circular dichroism spectroscopy.

Chromatography, Affinity↗

PubMed1995

Structure analysis of breast lesions using neighborhood graphs.

The College of American Pathologists divides proliferative lesions of the breast into three categories, but the diagnostic criteria for classification are not easy to apply. Therefore, many morphologic, immunohistochemical and morphometric features were studied in order to clarify this subdivision; however, the reported results are not very satisfactory. We also attempted to identify and differentiate the architectural features useful for the diagnosis of proliferative breast lesions. The morphologic characteristics of proliferative lesions of the breast were considered amenable to a graph theoretical approach. From a set of images of mammary ducts acquired from a hematoxylin-eosin-stained section, a hierarchy of graphs was built. The main graphs representing the structure are: (1) neighborhood graph, (2) planar graph, derived from (1) and (3) a dual graph. A simplified prototype of a system for structure analysis of the lesions was implemented and tested. Forty duct images classified by two expert pathologists were acquired and subjected to the system. The data obtained were analyzed, and some significant graph features validating our approach to the structure representation of the lesions were discovered. This approach can also be applied to other, similar classification problems and may help in understanding the biologic significance of proliferative lesions of the breast.

Breast Neoplasms↗

PubMed2003

Structuring parameters for assessment of bone quality using a morphological filter and star volume analysis: structuring property in the cancellous bone of the human femoral head.

This study examines structuring parameters for the assessment of bone quality based on the relationship of bone strength to the trabecular structure or bone mineral density (BMD). Thirty-nine human cancellous bone blocks sampled from resected Ward's triangle area were investigated. The trabecular bone pattern of each bone block was binarized into its corresponding trabecular skeletal pattern using computed radiography with a morphological filter. Every binarized trabecular skeletal pattern was quantified to find the trabecular skeletal pixel percentage (SkP = volume parameter of trabecular skeletal signal component) and skeletal star volume (Vt = connection parameter of trabecular skeletal structure). After BMD and elasticity for each bone block was measured by dual X-ray absorptiometry and the breaking test, the correlations to SkP and Vt were determined. In addition, an attempt was made to detect changes of the trabecular structure in the Ward's triangle area of the femoral head after patients had received treatment for osteoporosis. This evaluation used the structuring parameters SkP and Vt. The results showed that the larger the BMD value, the greater the elasticity, although some samples with similar BMD values had considerably different elasticities. With increased Vt values the trabecular skeletal pattern that had horizontal skeletal elements orientated perpendicular to the loading direction showed higher elasticity. No correlation between SkP and either elasticity or BMD was observed in either the subset (sequential images of morphological filter) or sumset images (combined with subset images). On the other hand, the correlation coefficient between Vt and elasticity was similar to that between BMD and elasticity for subset image ( n = 6). For sumset images (3-6 and 4-7), the correlation coefficient of Vt was equal or higher than that of BMD and the connectivity of the trabecular skeletal structure was more closely linked to bone strength as compared to BMD. Therefore, it was concluded that because no high correlation could be detected between Vt and BMD, BMD and the connectivity of trabeculae are independently related to bone strength. In addition, this method was used to evaluate patients after treatment for osteoporosis, and noticeable changes were seen in the bone trabecular skeletal structure in the Ward's triangle area of the femoral head. In reference to these clinical images, variations in SkP and Vt were quantitatively assessed. These results suggest that the SkP and Vt obtained using computed radiography, morphological filter, and star volume analysis are useful structuring parameters for the assessment of bone quality.

Aged↗

PubMed1987

Detailed structural analysis of exposed domains of membrane-bound Na+,K+-ATPase. A model of transmembrane arrangement.

Exposed regions of the alpha- and beta-subunits of membrane-bound Na+,K+-ATPase were in turn hydrolyzed with trypsin. Resistance of the beta-subunit to proteolysis was shown to be due mainly to the presence of disulfide bridge(s) in the molecule. A model for the spatial organisation of the enzyme in the membrane was proposed on the basis of detailed structural analysis of extramembrane regions of both subunits.

Amino Acid Sequence↗

PubMed2002

Tracking the putative biosynthetic precursors of oxygenated mycolates of Mycobacterium tuberculosis. Structural analysis of fatty acids of a mutant strain deviod of methoxy- and ketomycolates.

Disruption of the mma4 gene (renamed hma) of Mycobacterium tuberculosis has yielded a mutant strain defective in the synthesis of both keto- and methoxymycolates, with an altered cell-wall permeability to small molecules and a decreased virulence in the mouse model of infection (Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Lanéelle, M. A., Yu, K., Quémard, A., Smith, I., and Daffé, M. (2000) Mol. Microbiol. 36, 630-637). Assuming that the mutant would accumulate the putative precursors of the oxygenated mycolates of M. tuberculosis, a detailed structural analysis of mycolates from the hma-inactivated strain was performed using a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, proton NMR spectroscopy, and chemical degradation techniques. These consisted most exclusively of alpha-mycolates, composed of equal amounts of C(76)-C(82) dicyclopropanated (alpha(1)) and of C(77)-C(79) monoethylenic monocyclopropanated (alpha(2)) mycolates, the double bond being located at the "distal" position. In addition, small amounts of cis-epoxymycolates, structurally related to alpha(2)-mycolates, was produced by the mutant strain. Complementation of the hma-inactivated mutant with the wild-type gene resulted in the disappearance of the newly identified mycolates and the production of keto- and methoxymycolates of M. tuberculosis. Introduction of the hma gene in Mycobacterium smegmatis led to the lowering of diethylenic alpha mycolates of the recipient strain and the production of keto- and hydroxymycolates. These data indicate that long-chain ethylenic compounds may be the precursors of the oxygenated mycolates of M. tuberculosis. Because the lack of production of several methyltransferases involved in the biosynthesis of mycolates is known to decrease the virulence of the tubercle bacillus, the identification of the substrates of these enzymes should help in the design of inhibitors of the growth of M. tuberculosis.

Carbon↗

PubMed1982

A comparative structural analysis of the flagellin monomers of Caulobacter crescentus indicates that these proteins are encoded by two genes.

The flagellum of Caulobacter crescentus is composed of two flagellin polypeptide monomers which are distinguished by molecular weight and are closely related by biochemical and immunological criteria (C. Lagenaur and N. Agabian, J. Bacteriol. 132:731-733, 1977). The synthesis and assembly of these two flagellin proteins are developmentally regulated, and the periodicity of expression for each is distinct (C. Lagenaur and N. Agabian, J. Bacteriol. 135:1062-1069, 1978; M. A. Osley, M. Sheffery, and A. Newton, Cell 12:393-400, 1977). To understand the genetic and functional relationship between the 25,000- and 27,500-molecular-weight flagellins of C. crescentus CB15, a detailed comparative analysis of their protein structure was made, using a number of techniques, including one- and two-dimensional peptide mapping, a novel procedure of peptide alignment, and amino terminal amino acid sequence analysis. The tryptic peptides generated by each of the flagellins were compared by two-dimensional thin-layer chromatography. This peptide map analysis indicated that approximately 36% of the peptides generated from these two proteins had similar migration properties. Together with biochemical and immunological criteria, the two-dimensional peptide map suggested some structural relatedness between the monomers. However, a comparison of peptide fragments generated during partial protease digestion of each protein by a method of one-dimensional mapping indicated that the two proteins are structurally unique. A peptide alignment technique was developed to directly compare the primary structure of these proteins. In the peptide alignment procedure the amino terminus of each protein is radioactively labeled. After partial enzymatic digestion, the peptides are fractionated by polyacrylamide gel electrophoresis: those labeled at the amino terminus are then resolved by subsequent autoradiography. Each digest contains a family of amino-terminal-labeled fragments, the sizes of which reflect the sequential alignment of cleavage sites in the protein. A comparison of the alignment of specific cleavage sites of the two flagellins by this technique further established that each flagellin is structurally unique, particularly in the carboxyl terminal region. Finally, comparison of the amino terminal amino acid sequences indicated that the amino terminal region of both flagellins is highly conserved, but that the two polypeptides are clearly not identical. These findings strongly indicate that the two flagellins are encoded by distinct genetic loci and are not the product of novel processing of a single larger precursor.

Amino Acid Sequence↗

PubMed2002

Structural analysis of the inhibition of Cdk4 and Cdk6 by p16(INK4a) through molecular dynamics simulations.

Cyclin-dependent kinases 4, 6 and 2 (Cdk4/6/2), are proteins that lead progression through the G1-S transition, a step strictly regulated in the process of cell proliferation. The p16(INK4a) tumor suppressor, whose expression is inhibited in a high number of cancers, binds to Cdk4/6 and inhibits phosphorylation of the retinoblastoma protein, forcing cells to remain in the G1 phase and therefore, arresting cell division. Accordingly, the design of small compounds mimicking the inhibition of p16(INK4a) appears to be a promising way to treat cancer. In order to get some insight into the key interactions governing recognition between different cyclin-dependent kinases and the p16(INK4a) tumor suppressor, the present work reports the results of molecular dynamics simulations of both, the Cdk6-p16(INK4a) complex and the Cdk4-p16(INK4a) complex, respectively at 300 K. Most of the key interactions observed, were already anticipated in the analysis of the crystal structure of Cdk6-p16(INK4a). However, a few different features found out from the analysis of these calculations provide a better understanding of the role of the T-loop conformation, a fragment of Cdks, and the way the ATP binding-site is distorted upon binding of p16(INK4a).

Amino Acid Sequence↗

PubMedDate not supplied

Direct in situ structural analysis of recombinant outer membrane porins expressed in an OmpA-deficient mutant Escherichia coli strain.

An OmpA-deficient mutant of an OmpF/OmpC-free Escherichia coli B strain was selected using phage K3. The mutant strain was characterized by SDS-gel electrophoresis, immunoblotting, and electron microscopy. All major outer membrane proteins, including OmpA, were absent. This strain was then transformed with the plasmid pMY222 encoding the K12 OmpF porin or with pBlue-script-derived plasmids, encoding the porins OmpC, PhoE, and maltoporin, respectively. Following SDS extraction of outer membrane sacculi from strains expressing individual porins, crystalline porin arrays that allowed in situ structural analysis to be performed were observed. Furthermore, the absence of endogenous major outer membrane proteins facilitated the purification of native porin-lipopolysaccharide complexes, the functionally active channels, from the sacculi of transformed strains.

Bacterial Outer Membrane Proteins↗

PubMed2000

The lipocalin sperm coating lizard epididymal secretory protein family: mRNA structural analysis and sequential expression during the annual cycle of the lizard, Lacerta vivipara.

The epididymal epithelial cells of the lizard (Lacerta vivipara) produce large amounts of specific proteins under androgenic control. Amongst them, a major protein family that binds to the head of spermatozoa, the lizard epididymal secretory protein (LESP) family, has been identified as a member of the lipocalin superfamily. LESPs are composed of 9 elements that present an identical molecular mass of 18 000 Da but have a large range of pHi (3.5 to 9). The structural analysis of this protein family was performed by the determination and comparison of both the aminoterminal sequence of each element and the complete sequence of three specific LESP cDNA clones. When not identical, LESP elements present randomly dispatched nucleotide and amino acid substitutions, indicating the existence of at least five LESP mRNA populations encoded by a multigenic family. We determined that these LESP genes are differentially expressed during the annual epididymal cycle.

Amino Acid Sequence↗

PubMed1999

Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability.

The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.

Amino Acid Substitution↗

PubMed1991

Sequence and structural analysis of surface protein antigen I/II (SpaA) of Streptococcus sobrinus.

Streptococcal antigen I/II or the surface protein antigen A (SpaA) of Streptococcus sobrinus is an adhesin which mediates binding of the organism to tooth surfaces. The complete sequence of the gene which encodes SpaA has been determined. The gene consists of 4,584 bp and encodes a protein of 1,528 amino acid residues. The deduced amino acid sequence shows extensive homology with those of the cell surface adhesins from Streptococcus mutans serotypes c and f and from Streptococcus sanguis. Structural analysis of the N-terminal region (residues 50 to 550), which is rich in alanine and includes four tandem repeats of an 82-residue sequence, suggests that it adopts an alpha-helical coiled-coil conformation. Cell surface hydrophobicity may be associated with this region. The C-terminal region is more conserved and includes two tandem repeats of a 39-residue proline-rich sequence. A further proline-rich sequence in this region is predicted to span the cell wall. Although a hydrophobic sequence is present in the C-terminal region, it appears to be too short to span the cell membrane. Anchoring of SpaA in the cell membrane may therefore require some form of posttranslational modification or association with another membrane protein.

Adhesins, Bacterial↗

PubMed1982

The dream of Irma's injection: a structural analysis.

Lévi-Strauss developed a radically new, structural approach to the analysis of myth that may be adapted for use in the analysis of dreams. The method provides an alternative to psychoanalytic dream interpretation, focusing particularly on the internal dialectic of the dream--the movement from the initial premise to the resolution. This emphasis on the internal coherence and structured development of the dream contrasts strongly with the Freudian piecemeal decoding of dreams. The method is exemplified by analysis of Freud's classic Irma dream; it reveals a structure and message that have hitherto remained obscure. The interpretation connects the dream to Freud's theoretical ideas rather than to his supposed psychosexual fixations.

Association↗

PubMed2000

Structural analysis of diacyl peroxides by electrospray tandem mass spectrometry with ammonium acetate: bond homolysis of peroxide-ammonium and peroxide-proton adducts.

Organic peroxides have significant implications in organic chemistry and biological processes. The weak O-O bond makes them extremely difficult to characterize by conventional analytical methods. Diacyl peroxides are one of the major radical sources in polymerization and organic synthesis. It is well known that diacyl peroxides are thermal labile and thus are not amenable to study by gas chromatography/mass spectrometry (GC/MS). Electrospray tandem mass spectrometry (ESI-MS/MS) has been applied to the structural analysis of diacyl peroxides by formation of ammonium adducts. Collision induced dissociation (CID) studies of the ammonium adducts of the peroxide [M + NH(4)](+) give collision energy dependent fragments. For most diacyl peroxides, homolysis of the peroxy bond predominates the fragmentation pathways of the peroxide-ammonium adducts. Deuterated substrates have been employed to provide evidence for typical fragmentation pathways. The CID studies were also used to locate the O-18 in some O-18 specifically labeled diacyl peroxides. For branched alkyl or alkoxy substrates, McLafferty rearrangement and decarboxylation become a major pathway. By comparison with some anhydride analogues, ESI-MS/MS can also be used to study this class of compounds.

Mass Spectrometry↗

PubMed2003

Structural analysis of the functional influence of the surface peptide Gtf-P1 on Streptococcus mutans glucosyltransferase C activity.

Glucosyltransferases (GtfB/C/D) in Streptococcus mutans are responsible for synthesizing water-insoluble and water-soluble glucans from sucrose and play very crucial roles in the formation of dental plaque. A monoclonal antibody against a 19-mer peptide fragment named Gtf-P1 was found in GtfC to reduce the enzyme activity to 50%. However, a similar experiment suggested almost unchanged activity in GtfD, despite of the very high sequence homology between the two enzymes. No further details are yet available to elucidate the biochemical mechanism responsible for such discrimination. For a better understanding of the catalytic behavior of these glucosyltransferases, structural and functional analyses were performed. First, the exact epitope was identified to specify the residue(s) required for monoclonal antibody recognition. The results suggest that the discrimination is determined solely by single residue substitution. Second, based on a combined sequence and secondary structure alignment against known crystal structure of segments from closely related proteins, a three-dimensional homology model for GtfC was built. Structural analysis for the region communicating between Gtf-P1 and the catalytic triad revealed the possibility for an "en bloc" movement of hydrophobic residues, which may transduce the functional influence on enzyme activity from the surface of molecule into the proximity of the active site. Figure Side chain interactions between Gtf-P1 and catalytic Asp-477 in GtfC. Calpha-tracing of GtfC with the two crucial peptides (Gtf-P1, orange; Gtf-P2, blue) and the catalytic triad residues ( red) highlighted to show their relative spatial organization. Side chains for the residues are also depicted according to their atom types. The structure is viewed with the barrel opening facing down

Amino Acid Sequence↗

PubMed2001

Structural analysis of emerin, an inner nuclear membrane protein mutated in X-linked Emery-Dreifuss muscular dystrophy.

Like Duchenne and Becker muscular dystrophies, Emery-Dreifuss muscular dystrophy (EDMD) is characterized by myopathic and cardiomyopathic abnormalities. EDMD has the particularity of being linked to mutations in nuclear proteins. The X-linked form of EDMD is caused by mutations in the emerin gene, whereas autosomal dominant EDMD is caused by mutations in the lamin A/C gene. Emerin colocalizes with lamin A/C in interphase cells, and binds in vitro to lamin A/C. Recent work suggests that lamin A/C might serve as a receptor for emerin. We have undertaken a structural analysis of emerin, and in particular of its N-terminal domain, which is comprised in the emerin segment critical for binding to lamin A/C. We show that region 2-54 of emerin adopts the LEM fold. This fold was originally described in the two N-terminal domains of another inner nuclear membrane protein called lamina-associated protein 2 (LAP2). The existence of a conserved solvent-exposed surface on the LEM domains of LAP2 and emerin is discussed, as well as the nature of a possible common target.

Amino Acid Sequence↗

PubMed2001

Predicting the emergence of antibiotic resistance by directed evolution and structural analysis.

Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.

Amino Acid Sequence↗

PubMed1992

Mass spectrometry in the integrated strategy for the structural analysis of protein variants.

A significant proportion of genetic disorders are caused by point mutations in proteins, and many variants of haemoglobin have been characterized by mass spectrometry. The strategy employs fast atom bombardment and/or tandem mass spectrometry of enzymatically digested peptides for elucidating the type and position of the mutations. Electrospray ionization mass spectrometry is useful for detecting mutations in the intact protein molecule, but the competence diminishes with increasing mass or increasing number of 13C atoms. Recent developments of DNA techniques requires us to re-evaluate the status of mass spectrometry in this field. The role of mass spectrometry in the structural analysis of protein variants is discussed, and is compared with chemical methods and DNA strategies.

Amino Acid Sequence↗

PubMed1998

Structural analysis of the Spiroplasma virus, SpV4: implications for evolutionary variation to obtain host diversity among the Microviridae.

BACKGROUND: Spiroplasma virus, SpV4, is a small, non-enveloped virus that infects the helical mollicute Spiroplasma melliferum. SpV4 exhibits several similarities to the Chlamydia phage, Chp1, and the Coliphages alpha 3, phi K, G4 and phi X174. All of these viruses are members of the Microviridae. These viruses have isometric capsids with T = 1 icosahedral symmetry, cause lytic infections and are the only icosahedral phages that contain single-stranded circular DNA genomes. The aim of this comparative study on these phages was to understand the role of their capsid proteins during host receptor recognition. RESULTS: The three-dimensional structure of SpV4 was determined to 27 A resolution from images of frozen-hydrated particles. Cryo-electron microscopy (cryo-EM) revealed 20, approximately 54 A long, 'mushroom-like' protrusions on the surface of the capsid. Each protrusion comprises a trimeric structure that extends radially along the threefold icosahedral axes of the capsid. A 71 amino acid portion of VP1 (the SpV4 capsid protein) was shown, by structural alignment with the atomic structure of the F capsid protein of phi X174, to represent an insertion sequence between the E and F strands of the eight-stranded antiparallel beta-barrel. Secondary structure prediction of this insertion sequence provided the basis for a probable structural motif, consisting of a six-stranded antiparallel beta sheet connected by small turns. Three such motifs form the rigid stable trimeric structures (mushroom-like protrusions) at the threefold axes, with hydrophobic depressions at their distal surface. CONCLUSIONS: Sequence alignment and structural analysis indicate that distinct genera of the Microviridae might have evolved from a common primordial ancestor, with capsid surface variations, such as the SpV4 protrusions, resulting from gene fusion events that have enabled diverse host ranges. The hydrophobic nature of the cavity at the distal surface of the SpV4 protrusions suggests that this region may function as the receptor-recognition site during host infection.

Amino Acid Sequence↗