UNIPROT:Q86TM3 - FACTA Search

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Query: UNIPROT:Q86TM3 (cage)
29,987 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH(2)). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. (3)H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen egg white lysozyme (HEWL) and the monoclonal antibody IgG(1) D1.3. :CH(2) labeling of free HEWL or complexed with IgG(1) D1.3 yields 2.76 and 2.32 mmol CH(2) per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH(2). Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H(15)GLDNYR(21), G(117)TDVQAWIR(125), andG(22)YSLGNWVCAAK(33). Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.
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PMID:Exploring protein interfaces with a general photochemical reagent. 1660 Sep 65

The thermal unfolding pathway for dihydrolipoamide dehydrogenase (LipDH) isolated from Bacillus stearothermophilus was investigated focusing on the transient intermediate state characterized through time-resolved fluorescence studies. The decrease in ellipticity in the far UV region in the CD spectrum, the fluorescence spectral change of Trp-91 and FAD, and the thermal enzymatic inactivation curve consistently demonstrated that LipDH unfolded irreversibly on heat treatment at higher than 65 degrees C. LipDH took a transient intermediate state during the thermal unfolding process which could refold back into the native state. In this state, the internal rotation of FAD was activated in the polypeptide cage and correspondingly LipDH showed a peculiar conformation. The transient intermediate state of LipDH characterized in time-resolved fluorescence depolarization studies showed very similar properties to the molten-globule state, which has been confirmed in many studies on protein folding.
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PMID:Thermal unfolding process of dihydrolipoamide dehydrogenase studied by fluorescence spectroscopy. 1686 Dec 47

A new method of detecting the presence of deprotonation and determining its position in gas-phase polypeptide cations is described. The method involves 157-nm ultra-violet photodissociation (UVPD) and is based on monitoring the losses of CO2 (44 Da) from electronically excited deprotonated carboxylic groups relative to competing COOH losses (45 Da) from neutral carboxylic groups. Loss of CO2 is a strong indication of the presence of a zwitterionic [(+)...(-)...(+)] salt bridge in the gas-phase polypeptide cation. This method provides a tool for studying, for example, the nature of binding within polypeptide clusters. Collision-activated dissociation (CAD) of decarboxylated cations localizes the position of deprotonation. Fragment abundances can be used for the semiquantitative assessment of the branching ratio of deprotonation among different acidic sites, however, the mechanism of the fragment formation should be taken into account. Cations of Trp-cage proteins exist preferentially as zwitterions, with the deprotonation position divided between the Asp9 residue and the C terminus in the ratio 3:2. The majority of dications of the same molecule are not zwitterions. Furthermore, 157-nm UVPD produces abundant radical cations M*+ from protonated molecules through the loss of a hydrogen atom. This method of producing M*+ ions is general and can be applied to any gas-phase peptide cation. The abundance of the molecular radical cations M*+ produced is sufficient for further tandem mass spectrometry (MS/MS), which, in the cases studied, yielded side-chain loss of a basic amino acid as the most abundant fragmentation channel together with some backbone cleavages.
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PMID:Zwitterionic states in gas-phase polypeptide ions revealed by 157-nm ultra-violet photodissociation. 1687 5

The superfamily of ferritin-like proteins has recently expanded to include a phylogenetically distinct class of proteins termed DPS-like (DPSL) proteins. Despite their distinct genetic signatures, members of this subclass share considerable similarity to previously recognized DPS proteins. Like DPS, these proteins are expressed in response to oxidative stress, form dodecameric cage-like particles, preferentially utilize H(2)O(2) in the controlled oxidation of Fe(2+), and possess a short N-terminal extension implicated in stabilizing cellular DNA. Given these extensive similarities, the functional properties responsible for the preservation of the DPSL signature in the genomes of diverse prokaryotes have been unclear. Here, we describe the crystal structure of a DPSL protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Although the overall fold of the polypeptide chain and the oligomeric state of this protein are indistinguishable from those of authentic DPS proteins, several important differences are observed. First, rather than a ferroxidase site at the subunit interface, as is observed in all other DPS proteins, the ferroxidase site in SsDPSL is buried within the four-helix bundle, similar to bacterioferritin. Second, the structure reveals a channel leading from the exterior surface of SsDPSL to the bacterioferritin-like dimetal binding site, possibly allowing divalent cations and/or H(2)O(2) to access the active site. Third, a pair of cysteine residues unique to DPSL proteins is found adjacent to the dimetal binding site juxtaposed between the exterior surface of the protein and the active site channel. The cysteine residues in this thioferritin motif may play a redox active role, possibly serving to recycle iron at the ferroxidase center.
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PMID:Structure of the DPS-like protein from Sulfolobus solfataricus reveals a bacterioferritin-like dimetal binding site within a DPS-like dodecameric assembly. 1695 67

The alpha-helical coiled-coil motif serves as a robust scaffold for incorporating electron-transfer (ET) functionality into synthetic metalloproteins. These structures consist of a supercoiling of two or more aplha helices that are formed by the self-assembly of individual polypeptide chains whose sequences contain a repeating pattern of hydrophobic and hydrophilic residues. Early work from our group attached abiotic Ru-based redox sites to the most surface-exposed positions of two stranded coiled-coils and used electron-pulse radiolysis to study both intra- and intermolecular ET reactions in these systems. Later work used smaller metallopeptides to investigate the effects of conformational gating within electrostatic peptide-protein complexes. We have recently designed the C16C19-GGY peptide, which contains Cys residues located at both the "a" and "d" positions of its third heptad repeat in order to construct a nativelike metal-binding domain within its hydrophobic core. It was shown that the binding of both Cd(II) and Cu(I) ions induces the peptide to undergo a conformational change from a disordered random coil to a metal-bridged coiled-coil. However, whereas the Cd(II)-protein exists as a two-stranded coiled-coil, the Cu(I) derivative exists as a four-stranded coiled-coil. Upon the incorporation of other metal ions, metal-bridged peptide dimers, tetramers, and hexamers are formed. The Cu(I)-protein is of particular interest because it exhibits a long-lived (microsecond) room-temperature luminescence at 600 nm. The luminophore in this protein is thought to be a multinuclear CuI4Cys4(N/O)4 cage complex, which can be quenched by exogenous electron acceptors in solution, as shown by emission-lifetime and transient-absorption experiments. It is anticipated that further investigation into these systems will contribute to the expanding effort of bioinorganic chemists to prepare new kinds of functionally active synthetic metalloproteins.
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PMID:Incorporating electron-transfer functionality into synthetic metalloproteins from the bottom-up. 1714 Jan 93

The fragment molecular orbital method (FMO) has been used with a large number of wave functions for single-point calculations, and its high accuracy in comparison to ab initio methods has been well established. We have developed the analytic derivative of the electrostatic interaction between far separated fragments and performed a number of restricted Hartree-Fock (RHF) geometry optimizations using FMO and ab initio methods. In particular, the alpha-helix, beta-turn, and extended conformers of a 10-residue polyalanine were studied and the good FMO accuracy was established (the rms deviations for the former two forms were about 0.2 A and for the latter structure about 0.001 A). Met-enkephalin dimer was used as a model for the polypeptide binding and computed at the 3-21G and 6-31G* levels with a similar accuracy achieved; the error in the binding energy predictions (FMO vs ab initio) was 1-3 kcal/mol. Chignolin (PDB: 1uao) and an agonist polypeptide of the erythropoietin receptor protein (emp1) were optimized at the 3-21(+)G level, with the rms deviation from ab initio of about 0.2 A, or 0.5 degrees in terms of bond angles. The effect of solvation on the structure optimization was studied in chignolin and the Trp-cage miniprotein construct (PDB:1l2y), by describing water with TIP3P. The computed structures in gas phase and solution are compared to each other and experiment.
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PMID:The fragment molecular orbital method for geometry optimizations of polypeptides and proteins. 1738 63

Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations. Even though some unfolded and partially folded proteins are now known to possess biological function or to be involved in aggregation phenomena associated with disease states, experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a 'Trp-cage' miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR pulse-labelling experiments that involve rapid in situ protein refolding. These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.
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PMID:A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein. 1742 53

An extensive family of UDP-N-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferases (polypeptide N-acetylgalactosaminyltransferases, ppGalNAc-T's) catalyse the attachment of the first N-acetylgalactosamine (GalNAc) monosaccharide to the polypeptide at the initiation of O-linked glycosylation of proteins. Some members of the family are broadly expressed while others are more restricted in their distribution, their expression and activity being confined to certain cells or tissues, being associated with physiological states or differentiation. Their careful regulation, which is not well understood, may mediate the synthesis of varied glycoforms of cellular proteins with different biological activities. Disruptions in glycosylation are a common feature of cancer and may have functional significance. Immunocytochemistry with confocal scanning laser microscopy was employed to detect members of the ppGalNAc-T family, ppGalNAc-T1, -T2, -T3, -T4 and -T6 in a range of breast cell lines. The cells were chosen to represent a range of phenotypes from 'normal'/benign (HMT 3,522), primary, non-metastatic breast cancer (BT 474), to aggressive, metastatic breast cancer (ZR75-1, T47D, MCF-7, DU 4,475). They stably synthesise varying levels, consistent with origin and phenotype, of aberrantly glycosylated glycoproteins featuring exposed, terminal GalNAc residues, including the cancer-associated Tn antigen, which, in numerous studies, have been associated with metastatic competence and poor cancer prognosis. GalNAc-T1 and -T2 were detectable at low levels in all cell lines studied. ppGalNAc-T4, which has never been described in breast, was very weakly detectable in BT 474, MCF7 and T47D. ppGalNAc-T3 and -T6 were weakly detectable or undetectable, respectively, in the cell line HMT 3,522 derived from 'normal'/benign breast epithelium, but were readily detectable in all malignant cell lines. Thus, a broader range of ppGalNAc-T's were detectable in the malignant cell lines in comparison to the 'normal'/benign cells, where only the 'housekeeping' ppGalNAc-T1 and -T2 were present. Expression of normally tightly restricted ppGalNAc-T's may result in initiation of O-linked glycosylation at normally unoccupied potential glycosylation sites leading to altered glycoforms of proteins with changed biological activity which may contribute to the pathogenesis of cancer.
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PMID:Immunolocalisation of members of the polypeptide N-acetylgalactosaminyl transferase (ppGalNAc-T) family is consistent with biologically relevant altered cell surface glycosylation in breast cancer. 1744 26

Following the brief review of the modern fragment-based methods and other approaches to perform quantum-mechanical calculations of large systems, the theoretical development of the fragment molecular orbital method (FMO) is covered in detail, with the emphasis on the physical properties, which can be computed with FMO. The FMO-based polarizable continuum model (PCM) for treating the solvent effects in large systems and the pair interaction energy decomposition analysis (PIEDA) are described in some detail, and a range of applications of FMO to biological studies is introduced. The factors determining the relative stability of polypeptide conformers (alpha-helix, beta-turn, and extended form) are elucidated using FMO/PCM and PIEDA, and the interactions in the Trp-cage miniprotein construct (PDB: 1L2Y) are analyzed using PIEDA.
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PMID:Extending the power of quantum chemistry to large systems with the fragment molecular orbital method. 1751 37

Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a "triplex" protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N and C termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on the phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N and C termini facing the 2- and 3-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology.
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PMID:Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid. 1754 46

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