Relevant bibliographies by topics / Substrate-binding proteins

Academic literature on the topic 'Substrate-binding proteins'

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Published: 10 December 2022
Last updated: 29 January 2023

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Journal articles on the topic "Substrate-binding proteins"

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Berntsson, Ronnie P. A., Sander H. J. Smits, Lutz Schmitt, Dirk-Jan Slotboom, and Bert Poolman. "A structural classification of substrate-binding proteins." FEBS Letters 584, no. 12 (April 20, 2010): 2606–17. http://dx.doi.org/10.1016/j.febslet.2010.04.043.

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Scheepers, Giel H., Jelger A. Lycklama a Nijeholt, and Bert Poolman. "An updated structural classification of substrate-binding proteins." FEBS Letters 590, no. 23 (October 23, 2016): 4393–401. http://dx.doi.org/10.1002/1873-3468.12445.

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Manjeet, Kaur, Pallinti Purushotham, Chilukoti Neeraja, and Appa Rao Podile. "Bacterial chitin binding proteins show differential substrate binding and synergy with chitinases." Microbiological Research 168, no. 7 (August 2013): 461–68. http://dx.doi.org/10.1016/j.micres.2013.01.006.

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M. Counago, Rafael, Christopher A. McDevitt, Miranda P. Ween, and Bostjan Kobe. "Prokaryotic Substrate-Binding Proteins as Targets for Antimicrobial Therapies." Current Drug Targets 13, no. 11 (August 1, 2012): 1400–1410. http://dx.doi.org/10.2174/138945012803530170.

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Richarme, Gilbert, and Teresa Dantas Caldas. "Chaperone Properties of the Bacterial Periplasmic Substrate-binding Proteins." Journal of Biological Chemistry 272, no. 25 (June 20, 1997): 15607–12. http://dx.doi.org/10.1074/jbc.272.25.15607.

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Spooner, Paul J. R., W. John O’Reilly, Steven W. Homans, Nicholas G. Rutherford, Peter J. F. Henderson, and Anthony Watts. "Weak Substrate Binding to Transport Proteins Studied by NMR." Biophysical Journal 75, no. 6 (December 1998): 2794–800. http://dx.doi.org/10.1016/s0006-3495(98)77722-7.

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Pratt, R. F. "Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins)." Cellular and Molecular Life Sciences 65, no. 14 (April 14, 2008): 2138–55. http://dx.doi.org/10.1007/s00018-008-7591-7.

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Altenberg, Guillermo A. "The Engine of ABC Proteins." Physiology 18, no. 5 (October 2003): 191–95. http://dx.doi.org/10.1152/nips.01445.2003.

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Proteins that belong to the ATP-binding cassette superfamily span from bacteria to humans and comprise one of the largest protein families. These proteins are characterized by the presence of two nucleotide-binding domains, and recent studies suggest that association and dissociation of these domains is a common basic molecular mechanism of operation that couples ATP binding/hydrolysis to substrate transport across membranes.
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Schimpl, Marianne, Alexander W. Schüttelkopf, Vladimir S. Borodkin, and Daan M. F. van Aalten. "Human OGA binds substrates in a conserved peptide recognition groove." Biochemical Journal 432, no. 1 (October 25, 2010): 1–12. http://dx.doi.org/10.1042/bj20101338.

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Modification of cellular proteins with O-GlcNAc (O-linked N-acetylglucosamine) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and OGA (O-GlcNAcase or β-N-acetylglucosaminidase), which recognize their target proteins via as yet unidentified mechanisms. In the present study, we uncovered the first insights into the mechanism of substrate recognition by human OGA. The structure of a novel bacterial OGA orthologue reveals a putative substrate-binding groove, conserved in metazoan OGAs. Guided by this structure, conserved amino acids lining this groove in human OGA were mutated and the activity on three different substrate proteins [TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1), FoxO1 (forkhead box O1) and CREB (cAMP-response-element-binding protein)] was tested in an in vitro deglycosylation assay. The results provide the first evidence that human OGA may possess a substrate-recognition mechanism that involves interactions with O-GlcNAcylated proteins beyond the GlcNAc-binding site, with possible implications for differential regulation of cycling of O-GlcNAc on different proteins.
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Moutsita, R., J. Botti, MA Doyennette-Moyne, M. Aubery, and P. Codogno. "Cell spreading on laminin substrate involves Con A-binding proteins." Reproduction Nutrition Développement 30, no. 3 (1990): 397–401. http://dx.doi.org/10.1051/rnd:19900312.

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Dissertations / Theses on the topic "Substrate-binding proteins"

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Hendry, Garth S. "Dependence of substrate-water binding on protein and inorganic cofactors of photosystem II /." View thesis entry in Australian Digital Theses Program, 2002. http://thesis.anu.edu.au/public/adt-ANU20041124.140348/index.html.

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Fesser, Stephanie Marion. "Contribution of RNA binding proteins to substrate specificity in small RNA biogenesis." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-173105.

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Garza, John Anthony. "Structural and ligand-binding properties of a dual substrate specific enzymes from schizosaccharomyces pombe a dissertation /." San Antonio : UTHSC, 2009. http://learningobjects.library.uthscsa.edu/cdm4/item_viewer.php?CISOROOT=/theses&CISOPTR=45&CISOBOX=1&REC=17.

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Sarma, Ranjana. "Investigations of nucleotide-dependent electron transfer and substrate binding in nitrogen fixation and chlorophyll biosynthesis." Thesis, Montana State University, 2009. http://etd.lib.montana.edu/etd/2009/sarma/SarmaR1209.pdf.

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The studies presented in this thesis include studies of nucleotide-dependent conformations of the electron donor protein in nitrogenase and dark-operative protochlorophyllide reductase (DPOR) characterized using small-angle x-ray scattering and x-ray diffraction methods. Nitrogen fixation and chlorophyll synthesis are involved in the reduction of high energy bonds under physiological conditions. Both make use of elegant reaction mechanisms made possible by complex enzyme systems which are evolutionarily related. Nitrogenase reduces nitrogen to ammonia and is a two-component metalloenzyme composed of Fe protein and MoFe protein. For nitrogen reduction, the Fe protein and MoFe protein associate and dissociate in a manner concomitant with hydrolysis of at least two MgATP molecules and enables the concomitant transfer of at least one electron from Fe protein to MoFe protein. During chlorophyll biosysnthesis, the rate limiting step is catalyzed by a two-component metalloenzyme called DPOR. The two components of DPOR are BchL and BchNB proteins and these share high level of sequence similarity with the Fe protein and the MoFe protein, respectively. Based on this sequence similarity and biochemical data available, it is proposed that the reaction mechanism is similar to nitrogenase mechanism in which the components of DPOR associate and dissociate in a nucleotide dependent manner, to enable intercomponent electron transfer. Fe protein and BchL present as unique examples of proteins that couple nucleotide dependent conformational change to enable electron transfer for high energy bond reduction. The present studies have been directed at studying the low resolution studies of MgATP-bound wild-type Fe protein and its comparison to the structure of the proposed mimic, i.e, L127 Delta Fe protein. The studies presented show evidence of the MgATP-bound wild-type Fe protein having a conformation very different from the L127 Delta Fe protein. The chapters also include detailed characterization of the structure of BchL in both MgADP bound and nucleotide-free states which offer detailed insights in the structure based mechanism of BchL, with primary focus on identifying key residues involved in componenet docking and in electron transfer. Together, the studies on the Fe protein and BchL have furthered our understanding of mechanism of electron transfer in these complex enzyme systems.
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Jaya, Nomalie Naomi. "SUBSTRATE BINDING SITE FLEXIBILITY OF SMALL HEAT SHOCK PROTEINS AND FACTORS CONTRIBUTING TO EFFICIENT CHAPERONE ACTIVITY." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/193550.

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sHSPs maintain partially denaturing substrates in a soluble sHSP-substrate complex. The heterogeneous interaction between sHSPs and substrate within the complex has prevented a detailed study of the mechanism of sHSP substrate protection. Here, purified sHSPs and heat sensitive substrates were used to investigate the mechanism of sHSP chaperone action. Results presented provide new insights into how sHSPs recognize substrates, the architecture of the sHSP-substrate complex and factors contributing to chaperone efficiency.Direct evidence defining the role of the sHSP N-terminal arm and alpha crystallin domain in sHSP-substrate interactions is limited. A photoactivatable probe was site- specifically incorporated into PsHsp18.1, and cross-linking to substrate in sHSP-substrate complexes was quantified. The structurally flexible N-terminal arm of PsHsp18.1 makes strong contacts with both substrates tested, however differences in interaction were seen in the conserved alpha crystallin domain. Regions on the sHSP showing the strongest cross-links to substrates are buried within the dodecamer, supporting the model that the sHSP oligomer undergoes rearrangement or dissociation prior to substrate interactions.The arrangement of sHSPs and substrates whithin the complex is poorly defined. Limited proteolysis and chemical modification was combined with mass spectrometry to probe the sHSP-substrate complex using multiple sHSPs and substrates. This analysis reveals that a similar partially-denatured form of substrate is protected in the complex irrespective of sHSP identity. Further, sHSP in the complex is protected from proteolysis for a longer time compared to free sHSP. These data suggest that sHSPs and substrate are distributed both internally and on the periphery of the sHSP-substrate complex.Exact properties of the sHSP N-terminal arm contributing to protection are poorly defined. A molecular dynamics (MD) study was designed to test the hypothesis that the N-terminal arm could assume multiple conformations that can readily interact with denaturing substrates. Preliminary data suggest that at increased temperatures amino acids in the N-terminal arm form specific clusters which could act as substrate interaction sites. MD simulations, mutagenesis and altering the kinetics of substrate aggregation suggest that the conformational space occupied by the N-terminal arm at increased temperatures, along with flexibility and rate of substrate aggregation contribute to differences in chaperone efficiency.
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Hendry, Garth S., and Garth Hendry@baldwins com. "Dependence of substrate-water binding on protein and inorganic cofactors of photosystem II." The Australian National University. Research School of Biological Sciences, 2002. http://thesis.anu.edu.au./public/adt-ANU20041124.140348.

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The photosynthetic water oxidation reaction is catalyzed by an inorganic Mn4OxCaClyHCO3-z cluster at the heart of the oxygen evolving complex (OEC) in photosystem II. In the absence of an atomic resolution crystal structure, the precise molecular organization of the OEC remains unresolved. Accordingly, the role of the protein and inorganic cofactors of PSII (Ca2+, HCO3- and Cl-) in the mechanism of O2-evolution await clarification. In this study, rapid 18O-isotope exchange measurements were applied to monitor the substrate-water binding kinetics as a function of the intermediate S-states of the catalytic site (i.e. S3, S2 and S1) in Triton X-100 solubilized membrane preparations that are enriched in photosystem II activity and are routinely used to evaluate cofactor requirements. Consistent with the previous determinations of the 18O exchange behavior in thylakoids, the initial 18O exchange measurements of native PSII membranes at m/e = 34 (which is sensitive to the 16O18O product) show that the ‘fast’ and ‘slowly’ exchanging substrate-waters are bound to the catalytic site in the S3 state, immediately prior to O2 release. Although the slowly exchanging water is bound throughout the entire S-state cycle, the kinetics of the fast exchanging water remains too fast in the S2, S1 [and S0] states to be resolved using the current instrumentation, and left open the possibility that the second substrate-water only binds to the active site after the formation of the S3 state. Presented is the first direct evidence to show that fast exchanging water is already bound to the OEC in the S2 state. Rapid 18O-isotope exchange measurements for Ex-depleted PSII (depleted of the 17- and 23-kDa extrinsic proteins) in the S2 state reveals a resolvable fast kinetic component of 34k2 = 120 ± 14 s-1. The slowing down of the fast phase kinetics is discussed in terms of increased water permeation and the effect on the local dielectric following removal of the extrinsic subunits. In addition, the first direct evidence to show the involvement of calcium in substrate-water binding is also presented. Strontium replacement of the OEC Ca2+-site reveals a factor of ~3-4 increase in the 18O exchange of the slowly exchanging water across the S3, S2 and S1 states while the kinetics of the fast exchanging water remain unchanged. Finally, a re-investigation of the proposed role for bicarbonate as an oxidizable electron donor to photosystem II was unable to discern any 18O enrichment of the photosynthetically evolved O2 in the presence of 18O-bicarbonate. A working model for O2-evolution in terms of these results is presented.
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Fesser, Stephanie Marion [Verfasser], and Klaus [Akademischer Betreuer] Förstemann. "Contribution of RNA binding proteins to substrate specificity in small RNA biogenesis / Stephanie Marion Fesser. Betreuer: Klaus Förstemann." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/1055907793/34.

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Yuan, Ming. "Antiphagocytosis by Yersinia pseudotuberculosis : role of the YopH target proteins." Doctoral thesis, Umeå : Umeå University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-957.

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Wisniewska, Magdalena. "Biochemical studies on IGF and IGF-binding proteins interactions & structural investigations on the SH3 domain of Crk-associated tyrosine kinase substrate p130cas (CAS)." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=978198549.

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Escobedo, Pascual Albert. "Structural Insights into Substrate Binding and Regulation of E3 Ubiquitin Ligases in the Nedd4 Family using NMR Spectroscopy." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/284605.

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Nedd4L is a HECT-type E3 ubiquitin ligase (it covalently binds ubiquitin molecules before transferring them to the final substrate). Ubiquitination is a posttranslational modification (PTM) that labels proteins for a variety of fates, the most relevant one being proteasome-mediated degradation. Nedd4L is responsible for the regulation of the turnover of the sodium channel ß-ENaC as well as Smad2/3, mediator proteins of the signalling pathway activated by TGF-ß-like cytokines. It also targets the TGF-ß receptor itself. Defects in its function have been related to hereditary hypertension (Liddle’s syndrome), and could be relevant in certain sorts of cancer and metastasis. CDK8/9 and GSK3-ß are two kinases that regulate the phosphorylation of the Smads, enabling them to carry out their function in cooperation with transcription factors and other partner proteins. At the same time, they label the Smads for their recognition by ubiquitin ligases. This provides the cell with a mechanism to give a transient response to the cytokines of the TGF-ß type. In order to identify the residues and the phosphorylation patterns that are relevant for the interactions of the Smads with both the transcription factors and the ubiquitin ligases, we have prepared a set of phosphopeptides corresponding to the sequences of Smad1 and Smad3. Like all other members of the Nedd4 family, Nedd4L has a multi-domain architecture of the type C2-WW-HECT. Several ligases of the family exist in a latent conformation established through inter-domain contacts that occlude the catalytic site in the HECT domain, involving either the C2 domain (Smurf1, Smurf2, WWP2, Nedd4, Nedd4L) or the central segment where the WW domains are located (Itch). Certain cellular events displace these contacts, inducing the transition to the active conformation. In the case of Nedd4L, increases of the intracellular levels of Ca2+ activate the ligase. The hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) delivers into the cytosol the inositol 1,4,5-triphosphate (IP3), a second messenger that mobilizes the intracellular Ca2+ reserves. The C2 domain of Nedd4L interacts both with Ca2+ ions and with IP3. Using a structural and biophysical approach based on Nuclear Magnetic Resonance (NMR) we have described the specific interactions between the HECT and C2 domains that inhibit the catalytic function. Ca2+ binds the C2 domain with high affinity using the same binding surface and compromises these contacts. In addition, it mediates the interaction with IP3. These results provide the structural fundament for the activation and the relocation to the plasma membrane of Nedd4L mediated by Ca2+. The HECT domain has a highly conserved PY site (HECT-PY). The PY motifs are the sequences recognized by WW domains. Central to this recognition is the coordination of the tyrosine residue in the PY motif by the WW domain. In the crystallographic structure of the Nedd4L HECT domain the tyrosine residue of the HECT-PY motif appears buried in the hydrophobic core and not accessible for binding. It has been shown that the WW domains of Nedd4L recognize the HECT-PY motif of the ligase only after the unfolding of the HECT domain. We raised the hypothesis that the recognition of the HECT-PY motif by one of Nedd4L WW domains may play a role in the auto-ubiquitination mechanism of the ligase. Our data confirm that only when the fold of the HECT domain is partially damaged, the PY site is accessible for being recognized by the WW domains. We present the NMR solution structure of the complex between the WW3 domain and the HECT-PY motif. The site is protected in functional Nedd4L molecules, which are able to recognize it in damaged molecules and label them with ubiquitin for degradation.
Nedd4L és una E3 ubiquitín lligasa responsable de la regulació de la vida mitja del canal de sodi ß-ENaC i de Smad2/3, proteïnes mediadores de la ruta de senyalització activada per citocines TGF-ß. Defectes en la seva funció han estat relacionats amb la hipertensió hereditària (Síndrome de Liddle), i podrien ser rellevants en determinats tipus de càncer i metàstasi. CDK8/9 i GSK3-ß són dues quinases que regulen l’estat de fosforilació de les Smads, habilitant-les per dur a terme llur funció en cooperació amb factors de transcripció al mateix temps que les marquen per ser reconegudes per ubiquitín lligases. Amb l’objectiu d’identificar els residus i els patrons de fosforilació rellevants hem preparat un set de fosfopèptids que corresponen a les seqüències de Smad1/3. Nedd4L presenta una arquitectura multi-domini C2-WW-HECT. Diverses lligases de la família de Nedd4 existeixen en una conformació latent en què contactes inter-domini oclouen el lloc catalític en el domini HECT, involucrant bé el domini C2 (Smurf1/2, WWP2, Nedd4, Nedd4L) o la zona central amb els dominis WW (Itch). Certs esdeveniments cel•lulars desplacen aquests contactes, induint la transició a la conformació activa. L’increment dels nivells intracel•lulars de Ca2+ activa Nedd4L. La hidròlisi del fosfolípid de membrana PIP2 allibera l’IP3 provocant aquest increment. El domini C2 de Nedd4L interacciona tant amb el Ca2+ com amb l’IP3. Utilitzant l’RMN hem descrit els contactes HECT-C2 en la conformació latent i hem observat que el Ca2+ s’uneix al domini C2 amb alta afinitat utilitzant el mateix lloc d’unió, a més d’afavorir la interacció amb l’IP3. Així, hem aportat el fonament estructural per a l’activació i re­localització a la membrana cel•lular de Nedd4L. El domini HECT presenta un lloc PY altament conservat (HECT-PY). Els motius PY són reconeguts pels dominis WW. Proposem que el reconeixement del motiu HECT-PY per part d’un dels dominis WW de Nedd4L estigui implicat en l’auto-ubiquitinació. Hem observat que només quan el plegament del domini HECT està compromès, el lloc PY és accessible. Presentem l’estructura per RMN del complex WW3-HECT-PY. El motiu està protegit en molècules funcionals de Nedd4L, capaces de reconèixer-lo en molècules danyades i ubiquitinar-les.
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Book chapters on the topic "Substrate-binding proteins"

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Szegletes, Tivadar, William D. Mallender, and Terrone L. Rosenberry. "Substrate Binding to the Acetylcholinesterase Peripheral Site Promotes Substrate Hydrolysis but also Gives Rise to Substrate Inhibition." In Structure and Function of Cholinesterases and Related Proteins, 229. Boston, MA: Springer US, 1998. http://dx.doi.org/10.1007/978-1-4899-1540-5_59.

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Lee, Young Moo, Charles E. Larsen, and Jack Preiss. "Escherichia Coli ADPglucose Synthetase Substrate-Inhibitor Binding Sites Studied by Site(S) Directed Chemical Modification and Mutant Enzyme Characterization." In Proteins, 581–98. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4613-1787-6_58.

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Rosenberry, Terrone L., William D. Mallender, Patrick J. Thomas, and Tivadar Szegletes. "Substrate Binding to the Peripheral Site Occurs on the Catalytic Pathway of Acetylcholinesterase and Leads to Substrate Inhibition." In Structure and Function of Cholinesterases and Related Proteins, 189–96. Boston, MA: Springer US, 1998. http://dx.doi.org/10.1007/978-1-4899-1540-5_52.

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Mao, Youdong. "Structure, Dynamics and Function of the 26S Proteasome." In Subcellular Biochemistry, 1–151. Cham: Springer International Publishing, 2020. http://dx.doi.org/10.1007/978-3-030-58971-4_1.

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AbstractThe 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5 MDa mass, ubiquitously found in all eukaryotes. It selectively degrades ubiquitin-conjugated proteins and plays fundamentally indispensable roles in regulating almost all major aspects of cellular activities. To serve as the sole terminal “processor” for myriad ubiquitylation pathways, the proteasome evolved exceptional adaptability in dynamically organizing a large network of proteins, including ubiquitin receptors, shuttle factors, deubiquitinases, AAA-ATPase unfoldases, and ubiquitin ligases, to enable substrate selectivity and processing efficiency and to achieve regulation precision of a vast diversity of substrates. The inner working of the 26S proteasome is among the most sophisticated, enigmatic mechanisms of enzyme machinery in eukaryotic cells. Recent breakthroughs in three-dimensional atomic-level visualization of the 26S proteasome dynamics during polyubiquitylated substrate degradation elucidated an extensively detailed picture of its functional mechanisms, owing to progressive methodological advances associated with cryogenic electron microscopy (cryo-EM). Multiple sites of ubiquitin binding in the proteasome revealed a canonical mode of ubiquitin-dependent substrate engagement. The proteasome conformation in the act of substrate deubiquitylation provided insights into how the deubiquitylating activity of RPN11 is enhanced in the holoenzyme and is coupled to substrate translocation. Intriguingly, three principal modes of coordinated ATP hydrolysis in the heterohexameric AAA-ATPase motor were discovered to regulate intermediate functional steps of the proteasome, including ubiquitin-substrate engagement, deubiquitylation, initiation of substrate translocation and processive substrate degradation. The atomic dissection of the innermost working of the 26S proteasome opens up a new era in our understanding of the ubiquitin-proteasome system and has far-reaching implications in health and disease.
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Alberi, Tom, William A. Gilbert, Dagmar Ringe Ponzi, and Gregory A. Petsko. "The Role of Mobility in the Substrate Binding and Catalytic Machinery of Enzymes." In Ciba Foundation Symposium 93 - Mobility and Function in Proteins and Nucleic Acids, 4–24. Chichester, UK: John Wiley & Sons, Ltd., 2008. http://dx.doi.org/10.1002/9780470720752.ch2.

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Eichacker, L. A., M. Helfrich, W. Rüdiger, and B. Müller. "Substrate specificity studies of reaction center proteins P700, CP47, CP43, D2 and D1 for binding of chlorophyll." In Photosynthesis: from Light to Biosphere, 2925–28. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-009-0173-5_686.

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van Ophem, Peter W., Bryan W. Lepore, Kazuhisa Kishimoto, Dagmar Ringe, and James M. Manning. "Studies on an Active Site residue, E177, That Affects Binding of the Coenzyme in D-Amino Acid Transaminase, and Mechanistic Studies on a Suicide Substrate." In Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins, 339–46. Basel: Birkhäuser Basel, 2000. http://dx.doi.org/10.1007/978-3-0348-8397-9_56.

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Koonen, D. P. Y., W. A. Coumans, Y. Arumugam, A. Bonen, J. F. C. Glatz, and J. J. F. P. Luiken. "Giant membrane vesicles as a model to study cellular substrate uptake dissected from metabolism." In Cellular Lipid Binding Proteins, 121–30. Boston, MA: Springer US, 2002. http://dx.doi.org/10.1007/978-1-4419-9270-3_16.

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Gerke, Volker. "p11, A Member of the S-100 Protein Family, is Associated with the Tyrosine Kinase Substrate p36 (Annexin II)." In Novel Calcium-Binding Proteins, 139–55. Berlin, Heidelberg: Springer Berlin Heidelberg, 1991. http://dx.doi.org/10.1007/978-3-642-76150-8_9.

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da Graça Thrige, Dorthe, Jette Raun Byberg Buur, and Flemming Steen Jørgensen. "Substrate Binding and Catalytic Mechanism in Phospholipase C from Bacillus cereus." In Interacting Protein Domains, 93–96. Berlin, Heidelberg: Springer Berlin Heidelberg, 1997. http://dx.doi.org/10.1007/978-3-642-60848-3_15.

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Conference papers on the topic "Substrate-binding proteins"

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Liao, Jung-Chi, and George Oster. "The Engines of Biomolecular Motors." In ASME 2004 3rd Integrated Nanosystems Conference. ASMEDC, 2004. http://dx.doi.org/10.1115/nano2004-46094.

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The majority of biomolecular motors are powered by nucleoside triphosphate (NTP), especially adenosine triphosphate (ATP). These motors consist of a β-sheet with highly conserved motifs and the nucleotide binding domain around it. The highly conserved protein folds are the engines of these motors, which convert the energy of NTP hydrolysis cycle to mechanical work. Although functions of molecular motors are widely diverse, (including cargo movement, DNA unwinding, protein degradation, ion pumping, etc), the nucleotide binding domains are very similar. In the binding site, NTP undergoes a hydrolysis cycle E+NTP⇄E·NTP⇄E•NTP⇄E•NDP•Pi⇄E•NDP+Pi⇄E+NDP+Pi where E is the enzyme (motor protein), the small dot represents the docking of NTP, and the large dot represents the tightly-bound states. The hydrogen bond network formed in the NTP binding step, as shown in Figure 1 [1], deforms the β-sheet and adjacent structures. The local deformation propagates to conformational changes of functional residues to do mechanical work or to change the affinity to the substrate [2]. For multimeric motor proteins, we must also consider the stress paths among subunits which control the sequence and the activity of the protein. Stress trajectories emanating from a binding site either passes through a circumferential stress loop or a stress loop through the substrate.
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O'Brien, Peter J., Xiaolin Kang, Joseph Zachwieja, and John Lippincott. "Abstract 3195: Protein kinase Cβ substrate screens reveal enrichment for mRNA binding proteins and identify SERBP1 as a novel PKC substrate and signal transduction hub protein." In Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.am2013-3195.

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Jakobs, K. H., P. Gierschik, and R. Grandt. "THE ROLE OF GTP-BINDING PROTEINS EXHIBITING GTPase ACTIVITY IN PLATELET ACTIVATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644773.

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Activation of platelets by agonists acting via cell surface-located receptors apparently involves as an early event in transmembrane signalling an interaction of the agonist-occupied receptor with a guanine nucleotide-binding regulatory protein (G-protein). The activated G-protein, then, transduces the information to the effector molecule, being responsible for the changes in intracellular second messengers. At least two changes in intracellular signal molecules are often found to be associated with platelet activation by agonists, i.e., increases in inositol trisphosphate and diacylglycerol levels caused by activation of a polyphosphoinositide-specific phospholipase C and decrease in cyclic AMP concentration caused by inhibition of adenylate cyclase.Both actions of platelet-activating agents apparently involve G-proteins as transducing elements. Generally, the function of a G-protein in signal transduction can be measured either by its ability to regulate the activity of the effector molecule (phospholipase C or adenylate cyclase) or the binding affinity of an agonist to its specific receptor or by the abitlity of the G-protein to bind and hydrolyze GTP or one of its analogs in response to agonist-activated receptors. Some platelet-activating agonists (e.g. thrombin) can cause both adenylate cyclase inhibition and phospholipase C activation, whereas others induce either inhibition of adenylate cyclase (e.g. α2-adrenoceptor agonists) or activation of phospholipase C (e.g. stable endoperoxide analogs) . It is not yet known whether the simultaneous activation of two signal transduction systems is due to activation of two separate G-proteins by one receptor, to two distinct receptors activating each a distinct G-protein or to activation of two effector molecules by one G-protein.For some of the G-proteins, rather specific compounds are available causing inactivation of their function. In comparison to Gs, the stimulatory G-protein of the adenylate cyclase system, the adenylate cyclase inhibitory Gi-protein is rather specifically inactivated by ADP-ribosylation of its a-subunit by pertussis toxin, “unfortunately” not acting in intact platelets, and by SH-group reactive agents such as N-ethylmaleimide and diamide, apparently also affecting the Giα-subunit. Both of these treatments completely block α2-adrenoceptor-induced GTPase stimulation and adenylate cyclase inhibition and also thrombin-induced inhibition of adenylate cyclase. In order to know whether the G-protein coupling receptors to phospholipase C is similar to or different from the Gi-protein, high affinity GTPase stimulation by agents known to activate phospholipase C was evaluated in platelet membranes. The data obtained indicated that GTPase stimulation by agents causing both adenylate cyclase inhibition and phospholipase C activation is reduced, but only partially, by the above mentioned Gi-inactivating agents, while stimulation of GTPase by agents stimulating only phospholipase C is not affected by these treatments. These data suggested that the G-protein regulating phospholipase C activity in platelet membranes is different from the Gi-protein and may also not be a substrate for pertussis toxin. Measuring thrombin stimulation of inositol phosphate and diacylglycerol formation in saponin-permeabilized platelets, apparently contradictory data were reported after pertussis toxin treatment, being without effect or causing even an increase in thrombin stimulation of inositol phosphate formation (Lapetina: BBA 884, 219, 1986) or being inhibitory to thrombin stimulation of diacylglycerol formation (Brass et al.: JBC 261, 16838, 1986). These data indicate that the nature of the phospholipase C-related G-protein(s) is not yet defined and that their elucidation requires more specific tools as well as purification and reconstitution experiments. Preliminary data suggest that some antibiotics may serve as useful tools to characterize the phospho-lipase-related G-proteins. The possible role of G-protein phosphorylation by intracellular signal molecule-activated protein kinases in attenuation of signal transduction in platelets will be discussed.
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Tseng, F. G., H. M. Huang, C. S. Liu, C. Y. Huang, S. C. Lin, and C. C. Chieng. "Micro Protein Arrays Prepared by Microfabricated Stamps." In ASME 2000 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2000. http://dx.doi.org/10.1115/imece2000-1160.

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Abstract A novel protein arraying method is proposed by utilizing micro stamps to spot proteins on a bio-absorption surface. The method can pick up various protein solutions to spot onto a desired substrate for protein immobilization. The fabrication process of the micro stamp combines surface micromachining and molding process. Successful transfer of BSA protein solution onto a PVDF (Polyvinylidene difluoride) surface has been demonstrated by using the micro fabricated stamps. The size of stamped protein spot in this stage is about 20–50% larger than that of the stamp. To quickly verify the protein binding ability to two different substrates: PVDF and PhastGel® Pad, a chopstick system is used to pickup protein solution for stamping. Result shows that appreciable amount protein retention is achieved for at least 6 hours on both substrates. Improved spotting size and position control on micro stamping process has also been carried out by stamp surface coating with aluminum/aluminum oxide and stamp picking up protein-solutions from a protein-solution pre-wetted clean room tissue.
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Jovanović-Šanta, Suzana S., Aleksandar M. Oklješa, Antos B. Sachanka, Yaraslau U. Dzichenka, and Sergei A. Usanov. "17-SUBSTITUTED STEROIDAL TETRAZOLES – NOVEL LIGANDS FOR HUMAN STEROID-CONVERTING CYP ENZYMES." In 1st INTERNATIONAL Conference on Chemo and BioInformatics. Institute for Information Technologies, University of Kragujevac, 2021. http://dx.doi.org/10.46793/iccbi21.336js.

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In animal and human organisms, there are many enzymes, members of the family of heme- containing proteins, cytochromes P450 (CYPs), included in the biosynthesis and metabolism of many biomolecules, as cholesterol, bile acids, sex, and corticosteroid hormones, as well as in metabolism of drugs and xenobiotics. It is also well-known that different imidazole and triazole derivatives are efficient inhibitors of CYPs activity. In this study, we present in vitro screening of binding of novel androstane derivatives with tetrazole- containing substituents in position 17 to human recombinant steroid-converting CYP enzymes: CYP7A1, CYP7B1, CYP17A1, CYP19, and CYP21. Initial screening was performed using a high throughput screening approach, while the affinity of the ligands was analyzed using spectrophotometric titration. For some among tested compounds type I spectral response (substrate-like binding) for CYP7A1 selectively, while for one compound type II spectral response (inhibitor-like binding) for CYP21 were detected, with micromolar values of Kds. Interestingly, one compound with mixed spectral response was found to bind for CYP7B1, which means that there are two optimal positions of the ligand inside the protein active site. Such results could be useful in CYP-inhibiting drug development, during a fast, high-throughput screening of pharmacological potential of novel compounds, as well as in side- effects recognizing.
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Aakhus, A. M., N. O. Solum, and I. Hagen. "EFFECTS OF SOME ORGANIC SOLVENTS ON GP lb AND ACTIN-BINDING PROTEIN IN BLOOD PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643511.

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Effects on filamentous proteins appear to be a central phenomenon in the neuronal toxic effects of organic solvents. We have therefore compared the effects of some organic solvents (particularly isopropylalcohol, IPA) to the previously observed effects of dibucaine (DBC) on platelet cytoskeletal proteins. Incubation of platelets with 6% IPA at 37° C, like DBC, initiates a degradation of actin-binding protein (ABP) as substrate for a calcium activated protease (CAP), shown by SDS-PAGE. IPA leads to an increase followed by a decrease in bovine von Willebrand factor-induced agglutination. The decrease is accompanied by a release of glycocali-cin from the GP lb α-chain. The process was also studied using CIE of Triton X-100 extracts of platelets against antiserum to glycocalicin. Incubation of platelets with IPA before extraction in the presence of 4.2 mM leupeptin leads to a time-dependent transformation of GP Ib-related immunoprecipitates from that of the slow-migrating peak III complex (probably between ABP and GP lb) to the faster migrating GP Ib-precipitate. Our working hypothesis is that IPA induces an activation of the CAP by mobilizing calcium. This leads to degradation of ABP and liberation of GP lb from the cytoskeleton accompanied by an increased tendency for agglutination. The following decrease is explained by degradation of the glycocalicin part of the GP lb enchain which contains the binding-site for von Willebrand factor. We conclude that IPA has a similar effect on GP lb and ABP as DBC. Preliminary studies with 1% DMSO and 0,005% toluene at 37° C revealed that these organic solvents have some similar effects on platelets as described for IPA. Possibly the described effects are characteristic of certain cells at an early stage in a process ultimately leading to cell lysis.
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de Vries, C. J. M., N. K. Veerman, and H. Pannekoek. "ARTIFICIAL EXON SHUFFLING: CONSTRUCTION OF HYBRID cDNAS CONTAINING DOMAINS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (T-PA) AND UROKINASE (u-PA)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643940.

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The intriguing finding that functions of t-PA coincide with structural domains and that these domains occur in related proteins, has been the basis to construct hybrid proteins by artificial exon shuffling to prove the conservation of functions in the shuffled domains. The heavy chain (Hch) of t-PA mediates both binding to fibrin and stimulation of plasminogen activator activity via its Finger- and Kringle-2 domain, whereas the light chain (Lch) contains the serine protease moiety of the protein. The Hch of u-PA is very homologous to the Lch of t-PA, but exhibits a higher plasminogen activator activity. This activity of u-PA is not stimulated by fibrin. We employed the ‘M13 in vitro outlooping’ technique to fuse the Hch of t-PA cDNA and the Hch of u-PA cDNA, to create two different hybrid cDNAs. On one hybrid cDNA, the t-PA and the u-PA sequences are coupled precisely at the exon-intron boundaries of the corresponding genes, while the other hybrid cDNA lacks a u-PA segment at the junction, encoding 13 amino acids of u-PA. The hybrid cDNAs were transiently expressed in mouse Ltk- cells and the recombinant proteins were characterized. The plasminogen activator activity of these proteins was determined in an indirect amidolytic assay, using plasminogen and the chromogenic substrate S2251. As anticipated, the activity of both t-PA/u-PA hybrid proteins is stimulated by fibrin, however, not to the same extent as t-PA. Remarkably, we found a decreased inhibition of the hybrid proteins by the endothelial plasminogen activator inhibitor (PAI-1) as compared to t-PA and u-PA, although stable complexes between the hybrid proteins and the inhibitor are formed. We conclude that functions of structural domains are maintained during exon shuffling
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Rabiet, M. J., B. C. Furie, and B. Furie. "MOLECULAR DEFECT IN PROTHROMBIN MADRID: SUBSTITUTION OF ARGININE 273 BY CYSTEINE PRECLUDES ACTIVATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643936.

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Prothrombin Madrid, a mutant prothrombin, was detected in a patient with a excessive bleeding history. The defect was characterized by a low coagulant activity contrasting with a normal level of prothrombin antigen in plasma. Activation of the purified protein was impaired by the absence of one of the two factor Xa catalyzed cleavages, generating meizothrombin which expressed a thrombin-like activity but was inactive on fibrinogen (Guillin et al., Ann. N.Y. Acad. Sci. 370:414, 1981). Prothrombin and prothrombin Madrid were isolated directly from plasma, with high yield, by immunoaffinity chromatography using conformation specific antibodies immobilized on Sepharose. After reduction and alkylation, purified proteins were hydrolyzed by trypsin. Resulting peptides were separated by reverse phase HPLC. Comparison of the two peptide maps showed that the prothrombin Madrid digest contained an additional peptide, identified by automated Edman degradation as residues 269 to 287 in prothrombin with the substitution of cysteine for arginine at position 273. Peptide 274—287, present in the prothrombin digest, was missing in the prothrombin Madrid digest. The mutation, precluding cleavage by factor Xa and normal generation of thrombin, is identical to the one described for prothrombin Barcelona. The two patients families are not related, raising the possibility that the gene coding for the cysteine 273 mutation in prothrombin is more common than anticipated. Of the seven mutants of vitamin E-dependant blood clotting proteins structurally characterized to date, three are functionally defective due to the presence of the propeptide on the mature amino-ternfinus (factor IX Cambridge, Oxford 3 and San Dimas) and three are due to an alteration that precludes zymogen activation (faotor TX Chapel Hill, prothrombin Barcelona and Madrid). This sample remains too small to anticipate the different classes of point mutations seen in the human population but functional abnormalities of protein processing, metal and lipid binding, zymogen activation, substrate recognition and enzyme catalysis will likely be important phenotypes. However genetic defects may be limited to a discrete group of point mutations that have significant functional implication for the proteins
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Sarvestani, Alireza. "A Theoretical Analysis for the Effect of Substrate Elasticity on Cellular Adhesion." In ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology. ASMEDC, 2010. http://dx.doi.org/10.1115/nemb2010-13311.

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Cell behavior is mediated by variety of physiochemical properties of extracellular matrix (ECM). Material composition, surface chemistry, roughness, and distribution pattern of cell adhesive proteins are among the ECM properties which are known to modulate various cellular physiological functions. Mechanical stiffness of ECM in particular is found to be a major regulator for multiple aspects of cellular function. Experiments show that cells in general, exhibit an apparent adhesion preference for stiffer substrates with a larger projected spread area with increasing the substrate stiffness. In addition, it seems that the effect of substrates elasticity is strongly coupled with adhesivity of the substrate; on relatively stiff substrates the spread area of the cells exhibits strong biphasic dependence to the changes in ligand density, whereas on soft substrates their limited spreading is much less sensitive to the density of surface ligands. This study aims to propose a theoretical basis for the interplay between substrate elasticity and cellular adhesion, using an equilibrium thermodynamic model. Within this framework, the equilibrium contact area is assumed to ensure minimization of the free energy contributed by interfacial adhesive and repulsive interactions between the membrane and substrate as well as the deformation of cell and substrate. Hence, this thermodynamic model overlooks the contribution of intracellular signaling or actively regulated cytoskeleton and assumes that cell adhesion is solely a result of the balance between the membrane-substrate repulsive potentials, stored elastic energy, binding enthalpy, and mixing entropy of mobile receptors. The predictions of this purely mechanistic model for cell adhesion qualitatively follow the experimental results featuring the variation of cell spread area on compliant bio-adhesive substrates. This suggests that the mechanistic pathways inherent to membrane-substrate interactions may be equally important as intracellular signaling pathways to mediate the cellular adhesion.
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Baba, Waqas, and Sajid Maqsood. "Novel antihypertensive and anticholesterolemic peptides from peptic hydrolysates of camel whey proteins." In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/qecs2081.

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Hypercholesterolemia and hypertension are major growing concerns that are managed by drugs that inhibit various metabolic enzymes. Milk hydrolysates have been reported to contain various bioactive peptides (BAP) that can inhibit various metabolic enzymes for enhancing human health. As such camel whey proteins were subjected to peptic hydrolysis using a full factorial model (33) with hydrolysis time, temperature, and enzyme concentration as factors. The resulting hydrolysates were analyzed for anti-hypercholesterolemic and hypertensive properties by studying the in vitro inhibition of various enzymatic markers. The hydrolysates with lowest IC50 values were further subjected to LC-MS-QTOF that revealed presence of 185 peptides. Selected peptides that had Peptide Ranker Score greater than 0.8 were further studied for prediction of possible interactions with enzyme markers: pancreatic lipase (PL) cholesterol esterase (CE) and angiotensin converting enzyme (ACE) using in silico analysis. The data generated suggested that most of the peptides could bind active site of PL while as only three peptides could bind active site of CE. Based on higher number of reactive residues in the bioactive peptides (BAP) and greater number of substrate binding sites, FCCLGPVPP was identified as potential CE inhibitory peptide while PAGNFLPPVAAAPVM, MLPLMLPFTMGY, and LRFPL were identified as PL inhibitors. While peptides PAGNFLP, FCCLGPVPP, PAGNFLMNGLMHR, PAVACCLPPLPCHM were identified as potential ACE inhibitors. Molecular docking of selected peptides showed hydrophilic and hydrophobic interactions between peptides and target enzymes. Moreover, due to the importance of renin in managing hypertension, peptides from hydrolysates with high ACE inhibiting potential were predicted for potential to interact with renin using in silico analysis. Molecular docking was subsequently employed to identify how the identified peptides, PVAAAPVM and LRPFL, could interact with renin and potentially inhibit it. Thus, non-bovine (camel) whey hydrolysates might be used as functional ingredients for production of functional foods with antihypertensive and anticholesterolemic properties.
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Reports on the topic "Substrate-binding proteins"

1

Morrison, Mark, and Joshuah Miron. Molecular-Based Analysis of Cellulose Binding Proteins Involved with Adherence to Cellulose by Ruminococcus albus. United States Department of Agriculture, November 2000. http://dx.doi.org/10.32747/2000.7695844.bard.

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At the beginning of this project, it was clear that R. albus adhered tightly to cellulose and its efficient degradation of this polysaccharide was dependent on micromolar concentrations of phenylacetic acid (PAA) and phenylpropionic acid (PPA). The objectives for our research were: i) to identify how many different kinds of cellulose binding proteins are produced by Ruminococcus albus; ii) to isolate and clone the genes encoding some of these proteins from the same bacterium; iii) to determine where these various proteins were located and; iv) quantify the relative importance of these proteins in affecting the rate and extent to which the bacterium becomes attached to cellulose. BARD support has facilitated a number of breakthroughs relevant to our fundamental understanding of the adhesion process. First, R. albus possesses multiple mechanisms for adhesion to cellulose. The P.I.'s laboratory has discovered a novel cellulose-binding protein (CbpC) that belongs to the Pil-protein family, and in particular, the type 4 fimbrial proteins. We have also obtained genetic and biochemical evidence demonstrating that, in addition to CbpC-mediated adhesion, R. albus also produces a cellulosome-like complex for adhesion. These breakthroughs resulted from the isolation (in Israel and the US) of spontaneously arising mutants of R. albus strains SY3 and 8, which were completely or partially defective in adhesion to cellulose, respectively. While the SY3 mutant strain was incapable of growth with cellulose as the sole carbon source, the strain 8 mutants showed varying abilities to degrade and grow with cellulose. Biochemical and gene cloning experiments have been used in Israel and the US, respectively, to identify what are believed to be key components of a cellulosome. This combination of cellulose adhesion mechanisms has not been identified previously in any bacterium. Second, differential display, reverse transcription polymerase chain reaction (DD RT-PCR) has been developed for use with R. albus. A major limitation to cellulose research has been the intractability of cellulolytic bacteria to genetic manipulation by techniques such as transposon mutagenesis and gene displacement. The P.I.'s successfully developed DD RT- PCR, which expanded the scope of our research beyond the original objectives of the project, and a subset of the transcripts conditionally expressed in response to PAA and PPA have been identified and characterized. Third, proteins immunochemically related to the CbpC protein of R. albus 8 are present in other R. albus strains and F. intestinalis, Western immunoblots have been used to examine additional strains of R. albus, as well as other cellulolytic bacteria of ruminant origin, for production of proteins immunochemically related to the CbpC protein. The results of these experiments showed that R. albus strains SY3, 7 and B199 all possess a protein of ~25 kDa which cross-reacts with polyclonal anti-CbpC antiserum. Several strains of Butyrivibrio fibrisolvens, Ruminococcus flavefaciens strains C- 94 and FD-1, and Fibrobacter succinogenes S85 produced no proteins that cross-react with the same antiserum. Surprisingly though, F. intestinalis strain DR7 does possess a protein(s) of relatively large molecular mass (~200 kDa) that was strongly cross-reactive with the anti- CbpC antiserum. Scientifically, our studies have helped expand the scope of our fundamental understanding of adhesion mechanisms in cellulose-degrading bacteria, and validated the use of RNA-based techniques to examine physiological responses in bacteria that are nor amenable to genetic manipulations. Because efficient fiber hydrolysis by many anaerobic bacteria requires both tight adhesion to substrate and a stable cellulosome, we believe our findings are also the first step in providing the resources needed to achieve our long-term goal of increasing fiber digestibility in animals.
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Elbaum, Michael, and Peter J. Christie. Type IV Secretion System of Agrobacterium tumefaciens: Components and Structures. United States Department of Agriculture, March 2013. http://dx.doi.org/10.32747/2013.7699848.bard.

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Objectives: The overall goal of the project was to build an ultrastructural model of the Agrobacterium tumefaciens type IV secretion system (T4SS) based on electron microscopy, genetics, and immunolocalization of its components. There were four original aims: Aim 1: Define the contributions of contact-dependent and -independent plant signals to formation of novel morphological changes at the A. tumefaciens polar membrane. Aim 2: Genetic basis for morphological changes at the A. tumefaciens polar membrane. Aim 3: Immuno-localization of VirB proteins Aim 4: Structural definition of the substrate translocation route. There were no major revisions to the aims, and the work focused on the above questions. Background: Agrobacterium presents a unique example of inter-kingdom gene transfer. The process involves cell to cell transfer of both protein and DNA substrates via a contact-dependent mechanism akin to bacterial conjugation. Transfer is mediated by a T4SS. Intensive study of the Agrobacterium T4SS has made it an archetypal model for the genetics and biochemistry. The channel is assembled from eleven protein components encoded on the B operon in the virulence region of the tumor-inducing plasmid, plus an additional coupling protein, VirD4. During the course of our project two structural studies were published presenting X-ray crystallography and three-dimensional reconstruction from electron microscopy of a core complex of the channel assembled in vitro from homologous proteins of E. coli, representing VirB7, VirB9, and VirB10. Another study was published claiming that the secretion channels in Agrobacterium appear on helical arrays around the membrane perimeter and along the entire length of the bacterium. Helical arrangements in bacterial membranes have since fallen from favor however, and that finding was partially retracted in a second publication. Overall, the localization of the T4SS within the bacterial membranes remains enigmatic in the literature, and we believe that our results from this project make a significant advance. Summary of achievements : We found that polar inflations and other membrane disturbances relate to the activation conditions rather than to virulence protein expression. Activation requires low pH and nutrient-poor medium. These stress conditions are also reflected in DNA condensation to varying degrees. Nonetheless, they must be considered in modeling the T4SS as they represent the relevant conditions for its expression and activity. We identified the T4SS core component VirB7 at native expression levels using state of the art super-resolution light microscopy. This marker of the secretion system was found almost exclusively at the cell poles, and typically one pole. Immuno-electron microscopy identified the protein at the inner membrane, rather than at bridges across the inner and outer membranes. This suggests a rare or transient assembly of the secretion-competent channel, or alternatively a two-step secretion involving an intermediate step in the periplasmic space. We followed the expression of the major secreted effector, VirE2. This is a single-stranded DNA binding protein that forms a capsid around the transferred oligonucleotide, adapting the bacterial conjugation to the eukaryotic host. We found that over-expressed VirE2 forms filamentous complexes in the bacterial cytoplasm that could be observed both by conventional fluorescence microscopy and by correlative electron cryo-tomography. Using a non-retentive mutant we observed secretion of VirE2 from bacterial poles. We labeled the secreted substrates in vivo in order detect their secretion and appearance in the plant cells. However the low transfer efficiency and significant background signal have so far hampered this approach.
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