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PubMedDate not supplied

Modular, self-assembling peptide linkers for stable and regenerable carbon nanotube biosensor interfaces.

As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1 mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15 nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain.

Biosensing Techniques↗
PubMed2002

Abdominal imaging with a modular combination of spin and gradient echoes.

MR-CAT (combined acquisition technique), a modular hybrid imaging concept, was introduced recently. In this article it is demonstrated that the CAT principles can be applied to form a versatile combination of spin and gradient echoes for abdominal imaging. This CAT approach, which essentially integrates RARE and EPI modules in a sequential fashion, was used to implement a set of segmented and single-shot RARE/EPI-CAT imaging techniques. CAT was used in in vivo studies to perform high-resolution abdominal imaging in five healthy subjects. The results demonstrate the feasibility of abdominal imaging using the proposed CAT approach and the potential of this technique to reduce imaging time while preserving image quality.

Abdomen↗
PubMed1994

Modular arrangement of proteins as inferred from analysis of homology.

The structure of many proteins consists of a combination of discrete modules that have been shuffled during evolution. Such modules can frequently be recognized from the analysis of homology. Here we present a systematic analysis of the modular organization of all sequenced proteins. To achieve this we have developed an automatic method to identify protein domains from sequence comparisons. Homologous domains can then be clustered into consistent families. The method was applied to all 21,098 nonfragment protein sequences in SWISS-PROT 21.0, which was automatically reorganized into a comprehensive protein domain database, ProDom. We have constructed multiple sequence alignments for each domain family in ProDom, from which consensus sequences were generated. These nonreduntant domain consensuses are useful for fast homology searches. Domain organization in ProDom is exemplified for proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PEP:PTS) and for bacterial 2-component regulators. We provide 2 examples of previously unrecognized domain arrangements discovered with the help of ProDom.

Amino Acid Sequence↗
PubMed2003

Tool command language automation of the modular ion cyclotron data acquisition system (MIDAS) for data-dependent tandem Fourier transform ion cyclotron resonance mass spectrometry.

This manuscript describes the addition of data-dependent automation to the modular ion cyclotron resonance data acquisition system (MIDAS). The automation is made possible by developments and incorporation of a tool command language (Tcl) interpreter for automated acquisition. To accomplish the automation, real-time generation of excitation waveforms and scriptable data post-processing has been implemented into the MIDAS source code. In addition a new excitation event has also been added to allow for run-time generation of a single notch stored waveform inverse Fourier transform (SWIFT) excitation event. Examples of these new features and discussion of their enhancement to the existing data station are presented.

Adrenocorticotropic Hormone↗
PubMed2000

A modular set of prokaryotic and eukaryotic expression vectors.

A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine-GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae. In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed.

Amino Acid Sequence↗
PubMed1998

Modularity of prosody: autonomy of phonological quantity and intonation in aphasia.

The Finnish language is used here to enrich our conception of the dysprosody hypothesis of the output in Broca's agrammatism, i.e., of the claim that agrammatic speech is characterized by aberrant timing and intonation patterns. The native language of the two agrammatic aphasics of the present study places a high functional load on quantity (acoustically: timing/duration) in its phonology, as the short-long quantity opposition concerns both vowels and consonants. Moreover, the quantity opposition in Finnish is not local, i.e., syllable-internal as in many Germanic languages, because the phonetic quantity values are determined by a disyllabic sequence. In contrast to the high functional load of quantity in its lexical phonology, Finnish makes little, if any, grammatical use of intonation. In the present acoustic analyses it was noted that the two Broca's aphasics have preserved the disyllabically determined quantity oppositions, although the speech of the aphasics is characterized by long interword pauses, i.e., by syntactic dysprosody. In contrast to the asyntactic timing patterns, the declination line of intonation is retained in the output of the present aphasics in spite of the low grammatical value of intonation in the language. The present results further dispute the characterization of agrammatic speech output as being dysprosodic, or more specifically atemporal, as lexical quantity and syntactic tonal patterns can be retained in a speech mode that is replete with long interword pauses. More generally, the observations will corroborate the autonomy (modularity) of lexical and syntactic processes in oral language production.

Adult↗
PubMed1999

A retinoic acid-inducible modular protease in budding ascidians.

Retinoic acid-treated mesenchyme cells of the budding ascidian Polyandrocarpa misakiensis acquire an organizer activity to induce a secondary body axis when implanted into developing buds. We identified several different mRNAs that were upregulated in the mesenchyme cells after retinoic acid treatment. We isolated a cDNA clone corresponding to one of these mRNAs. The C-terminal region of the predicted protein product is homologous to the catalytic domain of serine proteases that belong to the trypsin family. The N-terminal region contains several types of protein-protein interaction domains. We therefore named this protein tunicate retinoic acid-inducible modular protease (TRAMP). Expression of the TRAMP mRNA in mesenchyme cells during budding and its upregulation by retinoic acid were demonstrated by reverse transcription-PCR and in situ hybridization. A glutathione S-transferase-TRAMP fusion protein showed a protease activity with trypsin-like substrate specificity and stimulated proliferation of the cell line established in this species.

Amino Acid Sequence↗
PubMed1999

A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites.

To construct large-insert libraries for the sequencing, mapping, and functional studies of complex genomes, we have constructed a new modular bacterial artificial chromosome (BAC) vector, pBACe3.6 (GenBank Accession No. U80929). This vector contains multiple cloning sites located within the sacB gene, allowing positive selection for recombinant clones on sucrose-containing medium. A recognition site for the PI-SceI nuclease has also been included, which permits linearization of recombinant DNA irrespective of the characteristics of the insert sequences. An attTn7 sequence present in pBACe3.6 permits retrofitting of BAC clones by Tn7-mediated insertion of desirable sequence elements into the vector portion. The ability to retrofit BAC clones will be useful for functional analysis of genes carried on the cloned inserts. The pBACe3.6 vector has been used for the construction of many genomic libraries currently serving as resources for large-scale mapping and sequencing.

Binding Sites↗
PubMed1995

The sarcin/ricin loop, a modular RNA.

The conformation of a 29 base oligonucleotide called E73 has been determined in solution by NMR. E73 includes a 23 nucleotide sequence that is identical with that of a the alpha-sarcin and ricin-sensitive loop (SRL) from rat 28 S rRNA, and like the SRL in intact ribosomes, E73 is a substrate for both toxins. The SRL includes a long, conserved sequence found in the RNA of all large ribosomal subunits, which plays a critical role in the factor-dependent steps of protein synthesis. The spectroscopic observations and analysis that led to the determination of the conformation of E73 are presented. The SRL in E73 has a highly structured conformation, which is stabilized by several non-Watson-Crick base-pairs, and many properties of the SRL in the ribosome can be understood assuming that the conformation of E73 and that of the SRL in the ribosome are the same. The role of the SRL in protein synthesis is discussed in light of the conformation of E73, as is the modular relationship that exists between the conformation of the SRL and other smaller RNAs.

Base Sequence↗
PubMed1996

The elastic I-band region of titin is assembled in a "modular" fashion by weakly interacting Ig-like domains.

The vertebrate striated muscle protein titin is thought to play a critical rôle in myofibril assembly and passive tension. The recently determined complete primary structure of titin revealed a modular architecture that opens the way to a structural characterisation and the understanding of essential properties of this molecule through dissection into units that are structurally and/or functionally relevant. To understand the assembly process of titin, and ultimately the molecular basis of its elastic behaviour, we studied the thermodynamic properties of module pairs, the smallest structural unit that includes a module-module interface. Thus, selected module pairs and their component single modules from the I-band part of the titin molecule were expressed in Escherichia coli and their heat-induced and denaturant-induced unfolding was investigated with a combination of techniques (circular dichroism, fluorescence spectroscopy and nuclear magnetic resonance). The stabilities of single modules and pairs were determined from denaturation experiments. The module interface was also modelled on the basis of the sequence alignment of all approximately 40 immunoglobulin like modules from the I-band and the known structure of one of them. Our results show that all modules and module pairs examined are independently folded in solution. When covalently linked, although weakly interacting, they still behave as autonomous co-operative units upon unfolding. These observations lead us to suggest that folding of titin in vitro is a hierarchical event and that weak interactions between its adjacent modules must only partly account for its presumed elastic function.

Amino Acid Sequence↗
PubMed1997

Modular structure of the trigger factor required for high activity in protein folding.

The Escherichia coli trigger factor is a peptidyl-prolyl cis/trans isomerase (PPIase) which catalyzes proline-limited protein folding extremely well. It has been found associated with nascent protein chains as well as with the chaperone GroEL. The trigger factor utilizes protein regions outside the central catalytic domain for catalyzing refolding of unfolded proteins efficiently. Here we produced several fragments which encompass individual domains or combinations of the middle FKBP-like domain (M) with the N-terminal (N) and C-terminal (C) regions, respectively. These fragments appear to be stably folded. They show ordered structure and cooperative urea-induced unfolding transitions, and the far-UV CD spectrum of the intact trigger factor is well represented by the sum of the spectra of the fragments. This suggests that the native trigger factor shows a modular structure, which is composed of three fairly independent folding units. In the intact protein there is a slight mutual stabilization of these units. The high enzymatic activity in protein folding could not be restored by fusing alternatively the N or the C-terminal regions to the catalytic domain (in NM and MC constructs, respectively). Surprisingly, the high folding activity of the intact trigger factor has been regained partially by functional complementation of the overlapping NM and MC constructs.

Amino Acid Isomerases↗
PubMed1997

Modular evolution of the respiratory NADH:ubiquinone oxidoreductase and the origin of its modules.

The proton-pumping NADH:ubiquinone oxidoreductase is the foremost of the respiratory chain complexes providing the proton motive force required for the synthesis of ATP. The complex is found in purple bacteria and in the mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits while the mitochondrial complex contains at least 28 accessory proteins which do not directly participate in the electron and proton transport function. A homologous complex which has 11 subunits in common with the respiratory complex I exists in cyanobacteria and chloroplasts. This complex might probably work as a NADPH:plastoquinone oxidoreductase being possibly involved in a cyclic photosynthetic electron transport. Homologues of the functional modules of the complex are also found in other bacterial electron transfer and ion transport proteins. The modular evolution of the complex and the possible origin of its modules are discussed in this paper.

Adenosine Triphosphate↗
PubMed2001

Biosynthesis of natural products on modular peptide synthetases.

Microbial nonribosomally processed peptides represent a large class of natural products including numerous important pharmaceutical agents, as well as other representatives that play a prevalent role in pathogenicity of certain microorganisms [M. A. Marahiel, T. Stachelhaus, and H. D. Mootz (1997). Chem. Rev. 97, 2651-2673]. Although diverse in structure, nonribosomally synthesized peptides have a common mode of biosynthesis. They are assembled on very large protein templates called peptide synthetases that exhibit a modular organization, allowing polymerization of monomers in an assembly-line-like mechanism.

Bacteria↗
PubMed2000

A modular set of helper-dependent herpes simplex virus expression vectors.

Herpes simplex virus (HSV) has many favorable properties in terms of its potential to serve as a delivery and expression platform for gene-based therapies, including the ability to establish persistent infections, a broad tissue tropism, episomal maintenance of transduced genes, and a large genome that can incorporate many additional cDNAs. Helper-dependent HSV vectors (commonly known as HSV amplicons) are well positioned to exploit the biology of the virus, since they contain only the two cis elements required for HSV replication and packaging and thus do not require the silencing of any viral genes to prevent toxicity to transduced cells over the course of cDNA expression. In this report we describe the development of a set of modular HSV amplicon vectors that can easily be modified to incorporate different genetic elements or alternatively can be used to retrofit existing expression constructs such that they can be packaged into infectious HSV particles.

Animals↗
PubMed2000

Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases.

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage.

Alcohol Dehydrogenase↗
PubMed2001

A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli.

To facilitate studies of multicomponent protein complexes, I have developed an Escherichia coli expression system which coexpresses up to four polypeptides from a single plasmid. The modular nature of the system enables efficient subcloning of a gene into each of the 4 cassettes in the polycistronic expression vector. Restriction sites present in the polycistronic expression vector allow both affinity tagged and untagged complexes to be overexpressed. I demonstrate successful use of the expression system for binary and ternary complexes, including the reconstitution of the VHL-elonginC-elonginB complex in E. coli and purification of the complex by affinity and ion-exchange chromatography. This polycistronic expression system should provide an important alternative to in vitro reconstitution of multicomponent complexes.

Base Sequence↗
PubMed1997

Reconstitution of a functional bovine papillomavirus type 1 origin of replication reveals a modular tripartite replicon with an essential AT-rich element.

A functional replication origin was reconstituted using oligonucleotide cassettes corresponding to three sequence subelements within the Bovine Papillomavirus Type 1 (BPV-1) replication origin: the 23-bp AT-rich region (ATR), the 18-bp binding site for the viral replication initiator protein E1 (E1BS), and a binding site for the viral transcriptional transactivator and replication enhancer protein E2 (E2BS). Replication of the reconstituted origin depended on heterologous expression of both the E1 and E2 proteins and on the presence of both the E1BS and E2BS, indicating that it is functionally analogous to the authentic BPV-1 origin. In addition, pairwise testing of subelement combinations revealed that the ATR was also essential and that a functional origin required at least one copy of all three subelements. While the E1BS and E2BS are sequence-specific elements, the function of the BPV-1 ATR could be at least partially substituted with heterologous AT-rich sequences, suggesting that the role of this element is primarily AT content-dependent rather than sequence-dependent. A stringent requirement for the ATR was also observed in the context of an authentic minimal origin sequence confirming that it is an intrinsic property of the BPV-1 origin and not simply an artifact of the reconstitution system. This study indicates that the minimal functional BPV-1 origin shares the tripartite modular organization characteristic of other simple eukaryotic replication origins. The reconstitution system described now provides a convenient approach to define the physical and functional interrelationships between the three subelements in a systematic fashion.

Adenovirus E2 Proteins↗
PubMed1998

Comparison of the lysogeny modules from the temperate Streptococcus thermophilus bacteriophages TP-J34 and Sfi21: implications for the modular theory of phage evolution.

A 7.6-kb DNA segment covering the putative lysogeny module of the pac-site-containing temperate Streptococcus thermophilus bacteriophage TP-J34 was sequenced. Sequence alignment with the lysogeny module from the cos-site-containing S. thermophilus bacteriophage phiSfi21 revealed areas of high sequence conservation (e.g., over the int gene), interspersed with regions of low or no sequence similarity (e.g., over the cro gene). Four of the six sharp transition zones from high to low sequence conservation were found within open reading frames coding for the CI repressor, the Anti-repressor, the Immunity protein, and a protein of unknown function. The transition points in the cI and ant genes appear to separate gene segments coding for distinct functional domains of these proteins. In addition, these two transition points were located at or near the deletion sites observed in spontaneous phage phiSfi21 deletion mutants, thus suggesting these transition points as recombinational hotspots. Furthermore, the sequence at the transition point in the cI gene resembles the attachment site of the phage, suggesting the involvement of the phage integrase in at least some of the exchange reactions. Contrary to the initial formulation of the modular theory of phage evolution the unit of the evolutionary exchange in streptococcal phages is not a group of functional genes, but can be as small as a single gene. Exchange reactions can also occur within genes, possibly between gene segments encoding distinct protein domains.

Amino Acid Sequence↗