Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle

2015-10-15

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM hasmore » several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 {angstrom} to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.« less

Biochemical characterization and structural insight into aliphatic β-amino acid adenylation enzymes IdnL1 and CmiS6.

PubMed

Cieślak, Jolanta; Miyanaga, Akimasa; Takaku, Ryoma; Takaishi, Makoto; Amagai, Keita; Kudo, Fumitaka; Eguchi, Tadashi

2017-07-01

Macrolactam antibiotics such as incednine and cremimycin possess an aliphatic β-amino acid as a starter unit of their polyketide chain. In the biosynthesis of incednine and cremimycin, unique stand-alone adenylation enzymes IdnL1 and CmiS6 select and activate the proper aliphatic β-amino acid as a starter unit. In this study, we describe the enzymatic characterization and the structural basis of substrate specificity of IdnL1 and CmiS6. Functional analysis revealed that IdnL1 and CmiS6 recognize 3-aminobutanoic acid and 3-aminononanoic acid, respectively. We solved the X-ray crystal structures of IdnL1 and CmiS6 to understand the recognition mechanism of these aliphatic β-amino acids. These structures revealed that IdnL1 and CmiS6 share a common recognition motif that interacts with the β-amino group of the substrates. However, the hydrophobic side-chains of the substrates are accommodated differently in the two enzymes. IdnL1 has a bulky Leu220 located close to the terminal methyl group of 3-aminobutanoate of the trapped acyl-adenylate intermediate to construct a shallow substrate-binding pocket. In contrast, CmiS6 possesses Gly220 at the corresponding position to accommodate 3-aminononanoic acid. This structural observation was supported by a mutational study. Thus, the size of amino acid residue at the 220 position is critical for the selection of an aliphatic β-amino acid substrate in these adenylation enzymes. Proteins 2017; 85:1238-1247. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

PubMed

Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

2013-04-05

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

Molecular recognition of pre-tRNA by Arabidopsis protein-only Ribonuclease P.

PubMed

Klemm, Bradley P; Karasik, Agnes; Kaitany, Kipchumba J; Shanmuganathan, Aranganathan; Henley, Matthew J; Thelen, Adam Z; Dewar, Allison J L; Jackson, Nathaniel D; Koutmos, Markos; Fierke, Carol A

2017-12-01

Protein-only ribonuclease P (PRORP) is an enzyme responsible for catalyzing the 5' end maturation of precursor transfer ribonucleic acids (pre-tRNAs) encoded by various cellular compartments in many eukaryotes. PRORPs from plants act as single-subunit enzymes and have been used as a model system for analyzing the function of the metazoan PRORP nuclease subunit, which requires two additional proteins for efficient catalysis. There are currently few molecular details known about the PRORP-pre-tRNA complex. Here, we characterize the determinants of substrate recognition by the single subunit Arabidopsis thaliana PRORP1 and PRORP2 using kinetic and thermodynamic experiments. The salt dependence of binding affinity suggests 4-5 contacts with backbone phosphodiester bonds on substrates, including a single phosphodiester contact with the pre-tRNA 5' leader, consistent with prior reports of short leader requirements. PRORPs contain an N-terminal pentatricopeptide repeat (PPR) domain, truncation of which results in a >30-fold decrease in substrate affinity. While most PPR-containing proteins have been implicated in single-stranded sequence-specific RNA recognition, we find that the PPR motifs of PRORPs recognize pre-tRNA substrates differently. Notably, the PPR domain residues most important for substrate binding in PRORPs do not correspond to positions involved in base recognition in other PPR proteins. Several of these residues are highly conserved in PRORPs from algae, plants, and metazoans, suggesting a conserved strategy for substrate recognition by the PRORP PPR domain. Furthermore, there is no evidence for sequence-specific interactions. This work clarifies molecular determinants of PRORP-substrate recognition and provides a new predictive model for the PRORP-substrate complex. © 2017 Klemm et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme.

PubMed

McCord, Lauren A; Liang, Wenguang G; Dowdell, Evan; Kalas, Vasilios; Hoey, Robert J; Koide, Akiko; Koide, Shohei; Tang, Wei-Jen

2013-08-20

Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ~18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

PubMed Central

Alderwick, Luke J.; Molle, Virginie; Kremer, Laurent; Cozzone, Alain J.; Dafforn, Timothy R.; Besra, Gurdyal S.; Fütterer, Klaus

2006-01-01

Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 Å), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 Å) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors. PMID:16477027

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

PubMed Central

Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

2013-01-01

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions.

PubMed

Feng, You; Maity, Ranjan; Whitelegge, Julian P; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T; Bedford, Mark T; Masson, Jean-Yves; Clarke, Steven G

2013-12-27

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F.

PubMed

Chen, Sheng; Wan, Hoi Ying

2011-01-15

BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions.

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

PubMed Central

Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

2013-01-01

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

Sortase Transpeptidases: Structural Biology and Catalytic Mechanism

PubMed Central

Jacobitz, Alex W.; Kattke, Michele D.; Wereszczynski, Jeff; Clubb, Robert T.

2017-01-01

Gram-positive bacteria use sortase cysteine transpeptidase enzymes to covalently attach proteins to their cell wall and to assemble pili. In pathogenic bacteria sortases are potential drug targets, as many of the proteins that they display on the microbial surface play key roles in the infection process. Moreover, the Staphylococcus aureus Sortase A (SaSrtA) enzyme has been developed into a valuable biochemical reagent because of its ability to ligate biomolecules together in vitro via a covalent peptide bond. Here we review what is known about the structures and catalytic mechanism of sortase enzymes. Based on their primary sequences, most sortase homologs can be classified into six distinct subfamilies, called class A–F enzymes. Atomic structures reveal unique, class-specific variations that support alternate substrate specificities, while structures of sortase enzymes bound to sorting signal mimics shed light onto the molecular basis of substrate recognition. The results of computational studies are reviewed that provide insight into how key reaction intermediates are stabilized during catalysis, as well as the mechanism and dynamics of substrate recognition. Lastly, the reported in vitro activities of sortases are compared, revealing that the transpeptidation activity of SaSrtA is at least 20-fold faster than other sortases that have thus far been characterized. Together, the results of the structural, computational, and biochemical studies discussed in this review begin to reveal how sortases decorate the microbial surface with proteins and pili, and may facilitate ongoing efforts to discover therapeutically useful small molecule inhibitors. PMID:28683919

Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor*

PubMed Central

Kobayashi, Hidetomo; Utsunomiya, Hiroko; Yamanaka, Hiroyasu; Sei, Yoshihisa; Katunuma, Nobuhiko; Okamoto, Keinosuke; Tsuge, Hideaki

2009-01-01

The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 Å resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs. PMID:19654332

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme

PubMed Central

Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P. C.; Ovaa, Huib; Drag, Marcin; Lima, Christopher D.; Huang, Tony T.

2015-01-01

Ubiquitin (Ub) and the ubiquitin-like modifier interferon stimulated gene 15 (ISG15) participate in the host defense of viral infections. Viruses, including the Severe Acute Respiratory Syndrome human coronavirus (SARS hCoV), have co-opted Ub/ISG15-conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub/ISG15-conjugated host proteins. Here, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle Eastern Respiratory Syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that similar to SARS PLpro, MERS PLpro is both a deubiquitinating and a deISGylating enzyme. Further analysis of the intrinsic deubiquitinating enzyme (DUB) activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, while SARS PLpro prefers to cleave Lys48-linked polyUb chains. Second, MERS PLpro cleaves polyUb chains in a “mono-distributive” manner (one Ub at a time), and SARS PLpro prefers to cleave K48-linked poly-Ub chains by sensing a di-Ub moiety as a minimal recognition element using a “di-distributive” cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses. PMID:25764917

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

PubMed

Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P C; Ovaa, Huib; Drag, Marcin; Lima, Christopher D; Huang, Tony T

2015-06-01

Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

PubMed Central

Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David

2009-01-01

Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays*

PubMed Central

Yu, Xiaobo; Woolery, Andrew R.; Luong, Phi; Hao, Yi Heng; Grammel, Markus; Westcott, Nathan; Park, Jin; Wang, Jie; Bian, Xiaofang; Demirkan, Gokhan; Hang, Howard C.; Orth, Kim; LaBaer, Joshua

2014-01-01

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications. PMID:25073739

The Fpg/Nei family of DNA glycosylases: substrates, structures, and search for damage.

PubMed

Prakash, Aishwarya; Doublié, Sylvie; Wallace, Susan S

2012-01-01

During the initial stages of the base excision DNA repair pathway, DNA glycosylases are responsible for locating and removing the majority of endogenous oxidative base lesions. The bifunctional formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of the Fpg/Nei family, one of the two families of glycosylases that recognize oxidized DNA bases, the other being the HhH/GPD (or Nth) superfamily. Structural and biochemical developments over the past decades have led to novel insights into the mechanism of damage recognition by the Fpg/Nei family of enzymes. Despite the overall structural similarity among members of this family, these enzymes exhibit distinct features that make them unique. This review summarizes the current structural knowledge of the Fpg/Nei family members, emphasizes their substrate specificities, and describes how these enzymes search for lesions. Copyright © 2012 Elsevier Inc. All rights reserved.

Catalyst recognition of cis-1,2-diols enables site-selective functionalization of complex molecules

NASA Astrophysics Data System (ADS)

Sun, Xixi; Lee, Hyelee; Lee, Sunggi; Tan, Kian L.

2013-09-01

Carbohydrates and natural products serve essential roles in nature, and also provide core scaffolds for pharmaceutical agents and vaccines. However, the inherent complexity of these molecules imposes significant synthetic hurdles for their selective functionalization and derivatization. Nature has, in part, addressed these issues by employing enzymes that are able to orient and activate substrates within a chiral pocket, which increases dramatically both the rate and selectivity of organic transformations. In this article we show that similar proximity effects can be utilized in the context of synthetic catalysts to achieve general and predictable site-selective functionalization of complex molecules. Unlike enzymes, our catalysts apply a single reversible covalent bond to recognize and bind to specific functional group displays within substrates. By combining this unique binding selectivity and asymmetric catalysis, we are able to modify the less reactive axial positions within monosaccharides and natural products.

Pattern Recognition of Adsorbing HP Lattice Proteins

NASA Astrophysics Data System (ADS)

Wilson, Matthew S.; Shi, Guangjie; Wüst, Thomas; Landau, David P.; Schmid, Friederike

2015-03-01

Protein adsorption is relevant in fields ranging from medicine to industry, and the qualitative behavior exhibited by course-grained models could shed insight for further research in such fields. Our study on the selective adsorption of lattice proteins utilizes the Wang-Landau algorithm to simulate the Hydrophobic-Polar (H-P) model with an efficient set of Monte Carlo moves. Each substrate is modeled as a square pattern of 9 lattice sites which attract either H or P monomers, and are located on an otherwise neutral surface. The fully enumerated set of 102 unique surfaces is simulated with each protein sequence. A collection of 27-monomer sequences is used- each of which is non-degenerate and protein-like. Thermodynamic quantities such as the specific heat and free energy are calculated from the density of states, and are used to investigate the adsorption of lattice proteins on patterned substrates. Research supported by NSF.

Functional group and stereochemical requirements for substrate binding by ghrelin O-acyltransferase revealed by unnatural amino acid incorporation.

PubMed

Cleverdon, Elizabeth R; Davis, Tasha R; Hougland, James L

2018-04-21

Ghrelin is a small peptide hormone that undergoes a unique posttranslational modification, serine octanoylation, to play its physiological roles in processes including hunger signaling and glucose metabolism. Ghrelin O-acyltransferase (GOAT) catalyzes this posttranslational modification, which is essential for ghrelin to bind and activate its cognate GHS-R1a receptor. Inhibition of GOAT offers a potential avenue for modulating ghrelin signaling for therapeutic effect. Defining the molecular characteristics of ghrelin that lead to binding and recognition by GOAT will facilitate the development and optimization of GOAT inhibitors. We show that small peptide mimics of ghrelin substituted with 2,3-diaminopropanoic acid in place of the serine at the site of octanoylation act as submicromolar inhibitors of GOAT. Using these chemically modified analogs of desacyl ghrelin, we define key functional groups within the N-terminal sequence of ghrelin essential for binding to GOAT and determine GOAT's tolerance to backbone methylations and altered amino acid stereochemistry within ghrelin. Our study provides a structure-activity analysis of ghrelin binding to GOAT that expands upon activity-based investigations of ghrelin recognition and establishes a new class of potent substrate-mimetic GOAT inhibitors for further investigation and therapeutic interventions targeting ghrelin signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

Crystal structure of lactose permease in complex with an affinity inactivator yields unique insight into sugar recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaptal, Vincent; Kwon, Seunghyug; Sawaya, Michael R.

Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl ofmore » the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.« less

Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.

2010-12-08

The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pocketsmore » that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.« less

Critical Determinants of Substrate Recognition by Cyclin-Dependent Kinase-like 5 (CDKL5).

PubMed

Katayama, Syouichi; Sueyoshi, Noriyuki; Kameshita, Isamu

2015-05-19

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase known to be associated with X-linked neurodevelopmental disorders. In a previous study, we identified amphiphysin 1 (Amph1) as a potential substrate for CDKL5 and identified a single phosphorylation site at Ser-293. In this study, we investigated the molecular mechanisms of substrate recognition by CDKL5 using Amph1 as a model substrate. Amph1 served as an efficient CDKL5 substrate, whereas Amph2, a structurally related homologue of Amph1, was not phosphorylated by CDKL5. The sequence around the Amph1 phosphorylation site is RPR(293)SPSQ, while the corresponding sequence in Amph2 is IPK(332)SPSQ. To define the amino acid sequence specificity of the substrate, various point mutants of Amph1 and Amph2 were prepared and phosphorylated by CDKL5. Both Amph2(I329R) and Amph1 served as efficient CDKL5 substrates, but Amph1(R290I) did not, indicating that the arginyl residue at the P -3 position is critical for substrate recognition. With regard to prolyl residues around the phosphorylation site of Amph1, Pro-291 at the P -2 position, but not Pro-294 at the P +1 position, is indispensable for phosphorylation by CDKL5. Phosphorylation experiments using various deletion mutants of Amph1 revealed that the proline-rich domain (PRD) (amino acids 247-315) alone was not phosphorylated by CDKL5. In contrast, Amph1(247-385), which comprised the PRD and CLAP domains, served as an efficient CDKL5 substrate. These results, taken together, suggest that both the phosphorylation site sequence (RPXSX) and the CLAP domain structure in Amph1 play crucial roles in recognition and phosphorylation by CDKL5.

The Calcineurin Signaling Network Evolves Via Conserved Kinase–Phosphatase Modules That Transcend Substrate Identity

PubMed Central

Bodenmiller, Bernd; Wanka, Stefanie; Landry, Christian R.; Aebersold, Ruedi; Cyert, Martha S.

2014-01-01

Summary To define the first functional network for calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase, we systematically identified its substrates in S. cerevisiae using phosphoproteomics and bioinformatics, followed by co-purification and dephosphorylation assays. This study establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution. Analyses of closely related yeasts show that many proteins were recently recruited to the network by acquiring a calcineurin-recognition motif. Calcineurin substrates in yeast and mammals are distinct due to network rewiring but surprisingly are phosphorylated by similar kinases. We postulate that co-recognition of conserved substrate features, including phosphorylation and docking motifs, preserves calcineurin-kinase opposition during evolution. One example we document is a composite docking site that confers substrate recognition by both calcineurin and MAPK. We propose that conserved kinase-phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop and establish common regulatory motifs in signaling networks. PMID:24930733

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

PubMed

Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

2008-09-05

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Critton, D.; Tortajada, A; Stetson, G

2008-01-01

Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPsmore » as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.« less

Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seongmin; Verdine, Gregory L.; Harvard)

2010-01-14

Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases havemore » been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.« less

Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.

PubMed

Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Wallnoefer, Hannes G; Liedl, Klaus R

2014-04-01

Proteases are prototypes of multispecific protein-protein interfaces. Proteases recognize and cleave protein and peptide substrates at a well-defined position in a substrate binding groove and a plethora of experimental techniques provide insights into their substrate recognition. We investigate the caspase family of cysteine proteases playing a key role in programmed cell death and inflammation, turning caspases into interesting drug targets. Specific ligand binding to one particular caspase is difficult to achieve, as substrate specificities of caspase isoforms are highly similar. In an effort to rationalize substrate specificity of two closely related caspases, we investigate the substrate promiscuity of the effector Caspases 3 and 7 by data mining (cleavage entropy) and by molecular dynamics simulations. We find a strong correlation between binding site rigidity and substrate readout for individual caspase subpockets explaining more stringent substrate readout of Caspase 7 via its narrower conformational space. Caspase 3 subpockets S3 and S4 show elevated local flexibility explaining the more unspecific substrate readout of that isoform in comparison to Caspase 7. We show by in silico exchange mutations in the S3 pocket of the proteases that a proline residue in Caspase 7 contributes to the narrowed conformational space of the binding site. These findings explain the substrate specificities of caspases via a mechanism of conformational selection and highlight the crucial importance of binding site local dynamics in substrate recognition of proteases. Proteins 2014; 82:546-555. © 2013 Wiley Periodicals, Inc. Copyright © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Structural insights into 5‧ flap DNA unwinding and incision by the human FAN1 dimer

NASA Astrophysics Data System (ADS)

Zhao, Qi; Xue, Xiaoyu; Longerich, Simonne; Sung, Patrick; Xiong, Yong

2014-12-01

Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 5‧ flap DNA substrate, showing that two FAN1 molecules form a head-to-tail dimer to locate the lesion, orient the DNA and unwind a 5‧ flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1.

The Diversity of Ribonuclease P: Protein and RNA Catalysts with Analogous Biological Functions

PubMed Central

Klemm, Bradley P.; Wu, Nancy; Chen, Yu; Liu, Xin; Kaitany, Kipchumba J.; Howard, Michael J.; Fierke, Carol A.

2016-01-01

Ribonuclease P (RNase P) is an essential endonuclease responsible for catalyzing 5’ end maturation in precursor transfer RNAs. Since its discovery in the 1970s, RNase P enzymes have been identified and studied throughout the three domains of life. Interestingly, RNase P is either RNA-based, with a catalytic RNA subunit, or a protein-only (PRORP) enzyme with differential evolutionary distribution. The available structural data, including the active site data, provides insight into catalysis and substrate recognition. The hydrolytic and kinetic mechanisms of the two forms of RNase P enzymes are similar, yet features unique to the RNA-based and PRORP enzymes are consistent with different evolutionary origins. The various RNase P enzymes, in addition to their primary role in tRNA 5’ maturation, catalyze cleavage of a variety of alternative substrates, indicating a diversification of RNase P function in vivo. The review concludes with a discussion of recent advances and interesting research directions in the field. PMID:27187488

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.

PubMed

Fuchs, Julian E; Huber, Roland G; Waldner, Birgit J; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R

2015-01-01

Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.

A cell wall-degrading esterase of Xanthomonas oryzae requires a unique substrate recognition module for pathogenesis on rice.

PubMed

Aparna, Gudlur; Chatterjee, Avradip; Sonti, Ramesh V; Sankaranarayanan, Rajan

2009-06-01

Xanthomonas oryzae pv oryzae (Xoo) causes bacterial blight, a serious disease of rice (Oryza sativa). LipA is a secretory virulence factor of Xoo, implicated in degradation of rice cell walls and the concomitant elicitation of innate immune responses, such as callose deposition and programmed cell death. Here, we present the high-resolution structural characterization of LipA that reveals an all-helical ligand binding module as a distinct functional attachment to the canonical hydrolase catalytic domain. We demonstrate that the enzyme binds to a glycoside ligand through a rigid pocket comprising distinct carbohydrate-specific and acyl chain recognition sites where the catalytic triad is situated 15 A from the anchored carbohydrate. Point mutations disrupting the carbohydrate anchor site or blocking the pocket, even at a considerable distance from the enzyme active site, can abrogate in planta LipA function, exemplified by loss of both virulence and the ability to elicit host defense responses. A high conservation of the module across genus Xanthomonas emphasizes the significance of this unique plant cell wall-degrading function for this important group of plant pathogenic bacteria. A comparison with the related structural families illustrates how a typical lipase is recruited to act on plant cell walls to promote virulence, thus providing a remarkable example of the emergence of novel functions around existing scaffolds for increased proficiency of pathogenesis during pathogen-plant coevolution.

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

PubMed Central

Fuchs, Julian E.; Huber, Roland G.; Waldner, Birgit J.; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R.

2015-01-01

Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm “dynamics govern specificity” might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design. PMID:26496636

The Crystal Structure Analysis of Group B Streptococcus Sortase C1: A Model for the ;Lid; Movement upon Substrate Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khare, Baldeep; Fu, Zheng-Qing; Huang, I-Hsiu

2012-02-07

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible 'lid' anchored in the active site. However, the mechanistic details of the 'lid' displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its 'lid' mutant and proposed a role of the 'lid' as a protectormore » of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.« less

Adolescents' ability to read different emotional faces relates to their history of maltreatment and type of psychopathology.

PubMed

Leist, Tatyana; Dadds, Mark R

2009-04-01

Emotional processing styles appear to characterize various forms of psychopathology and environmental adversity in children. For example, autistic, anxious, high- and low-emotion conduct problem children, and children who have been maltreated, all appear to show specific deficits and strengths in recognizing the facial expressions of emotions. Until now, the relationships between emotion recognition, antisocial behaviour, emotional problems, callous-unemotional (CU) traits and early maltreatment have never been assessed simultaneously in one study, and the specific associations of emotion recognition to maltreatment and child characteristics are therefore unknown. We examined facial-emotion processing in a sample of 23 adolescents selected for high-risk status on the variables of interest. As expected, maltreatment and child characteristics showed unique associations. CU traits were uniquely related to impairments in fear recognition. Antisocial behaviour was uniquely associated with better fear recognition, but impaired anger recognition. Emotional problems were associated with better recognition of anger and sadness, but lower recognition of neutral faces. Maltreatment was predictive of superior recognition of fear and sadness. The findings are considered in terms of social information-processing theories of psychopathology. Implications for clinical interventions are discussed.

Face recognition in the thermal infrared domain

NASA Astrophysics Data System (ADS)

Kowalski, M.; Grudzień, A.; Palka, N.; Szustakowski, M.

2017-10-01

Biometrics refers to unique human characteristics. Each unique characteristic may be used to label and describe individuals and for automatic recognition of a person based on physiological or behavioural properties. One of the most natural and the most popular biometric trait is a face. The most common research methods on face recognition are based on visible light. State-of-the-art face recognition systems operating in the visible light spectrum achieve very high level of recognition accuracy under controlled environmental conditions. Thermal infrared imagery seems to be a promising alternative or complement to visible range imaging due to its relatively high resistance to illumination changes. A thermal infrared image of the human face presents its unique heat-signature and can be used for recognition. The characteristics of thermal images maintain advantages over visible light images, and can be used to improve algorithms of human face recognition in several aspects. Mid-wavelength or far-wavelength infrared also referred to as thermal infrared seems to be promising alternatives. We present the study on 1:1 recognition in thermal infrared domain. The two approaches we are considering are stand-off face verification of non-moving person as well as stop-less face verification on-the-move. The paper presents methodology of our studies and challenges for face recognition systems in the thermal infrared domain.

Recognition of facial emotions in neuropsychiatric disorders.

PubMed

Kohler, Christian G; Turner, Travis H; Gur, Raquel E; Gur, Ruben C

2004-04-01

Recognition of facial emotions represents an important aspect of interpersonal communication and is governed by select neural substrates. We present data on emotion recognition in healthy young adults utilizing a novel set of color photographs of evoked universal emotions. In addition, we review the recent literature on emotion recognition in psychiatric and neurologic disorders, and studies that compare different disorders.

Intraspecific Variation in Learning: Worker Wasps Are Less Able to Learn and Remember Individual Conspecific Faces than Queen Wasps.

PubMed

Tibbetts, Elizabeth A; Injaian, Allison; Sheehan, Michael J; Desjardins, Nicole

2018-05-01

Research on individual recognition often focuses on species-typical recognition abilities rather than assessing intraspecific variation in recognition. As individual recognition is cognitively costly, the capacity for recognition may vary within species. We test how individual face recognition differs between nest-founding queens (foundresses) and workers in Polistes fuscatus paper wasps. Individual recognition mediates dominance interactions among foundresses. Three previously published experiments have shown that foundresses (1) benefit by advertising their identity with distinctive facial patterns that facilitate recognition, (2) have robust memories of individuals, and (3) rapidly learn to distinguish between face images. Like foundresses, workers have variable facial patterns and are capable of individual recognition. However, worker dominance interactions are muted. Therefore, individual recognition may be less important for workers than for foundresses. We find that (1) workers with unique faces receive amounts of aggression similar to those of workers with common faces, indicating that wasps do not benefit from advertising their individual identity with a unique appearance; (2) workers lack robust memories for individuals, as they cannot remember unique conspecifics after a 6-day separation; and (3) workers learn to distinguish between facial images more slowly than foundresses during training. The recognition differences between foundresses and workers are notable because Polistes lack discrete castes; foundresses and workers are morphologically similar, and workers can take over as queens. Overall, social benefits and receiver capacity for individual recognition are surprisingly plastic.

Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependent C-methylation.

PubMed

Ariyawutthiphan, Orapin; Ose, Toyoyuki; Minami, Atsushi; Shinde, Sandip; Sinde, Sandip; Tsuda, Muneya; Gao, Yong-Gui; Yao, Min; Oikawa, Hideaki; Tanaka, Isao

2012-11-01

In the typical isoprenoid-biosynthesis pathway, condensation of the universal C(5)-unit precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) occurs via the common intermediates prenyl pyrophosphates (C(10)-C(20)). The diversity of isoprenoids reflects differences in chain length, cyclization and further additional modification after cyclization. In contrast, the biosynthesis of 2-methylisonorneol (2-MIB), which is responsible for taste and odour problems in drinking water, is unique in that it primes the enzymatic methylation of geranyl pyrophosphate (GPP) before cyclization, which is catalyzed by an S-adenosyl-L-methionine-dependent methyltransferase (GPPMT). The substrate of GPPMT contains a nonconjugated olefin and the reaction mechanism is expected to be similar to that of the steroid methyltransferase (SMT) family. Here, structural analysis of GPPMT in complex with its cofactor and substrate revealed the mechanisms of substrate recognition and possible enzymatic reaction. Using the structures of these complexes, methyl-group transfer and the subsequent proton-abstraction mechanism are discussed. GPPMT and SMTs contain a conserved glutamate residue that is likely to play a role as a general base. Comparison with the reaction mechanism of the mycolic acid cyclopropane synthase (MACS) family also supports this result. This enzyme represented here is the first model of the enzymatic C-methylation of a nonconjugated olefin in the isoprenoid-biosynthesis pathway. In addition, an elaborate system to avoid methylation of incorrect substrates is proposed.

Large-Scale, Lineage-Specific Expansion of a Bric-a-Brac/Tramtrack/Broad Complex Ubiquitin-Ligase Gene Family in Rice[W

PubMed Central

Gingerich, Derek J.; Hanada, Kousuke; Shiu, Shin-Han; Vierstra, Richard D.

2007-01-01

Selective ubiquitination of proteins is directed by diverse families of ubiquitin-protein ligases (or E3s) in plants. One important type uses Cullin-3 as a scaffold to assemble multisubunit E3 complexes containing one of a multitude of bric-a-brac/tramtrack/broad complex (BTB) proteins that function as substrate recognition factors. We previously described the 80-member BTB gene superfamily in Arabidopsis thaliana. Here, we describe the complete BTB superfamily in rice (Oryza sativa spp japonica cv Nipponbare) that contains 149 BTB domain–encoding genes and 43 putative pseudogenes. Amino acid sequence comparisons of the rice and Arabidopsis superfamilies revealed a near equal repertoire of putative substrate recognition module types. However, phylogenetic comparisons detected numerous gene duplication and/or loss events since the rice and Arabidopsis BTB lineages split, suggesting possible functional specialization within individual BTB families. In particular, a major expansion and diversification of a subset of BTB proteins containing Meprin and TRAF homology (MATH) substrate recognition sites was evident in rice and other monocots that likely occurred following the monocot/dicot split. The MATH domain of a subset appears to have evolved significantly faster than those in a smaller core subset that predates flowering plants, suggesting that the substrate recognition module in many monocot MATH-BTB E3s are diversifying to ubiquitinate a set of substrates that are themselves rapidly changing. Intriguing possibilities include pathogen proteins attempting to avoid inactivation by the monocot host. PMID:17720868

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

PubMed

Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

2012-07-01

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

A Look Inside HIV Resistance through Retroviral Protease Interaction Maps

PubMed Central

Kontijevskis, Aleksejs; Prusis, Peteris; Petrovska, Ramona; Yahorava, Sviatlana; Mutulis, Felikss; Mutule, Ilze; Komorowski, Jan; Wikberg, Jarl E. S

2007-01-01

Retroviruses affect a large number of species, from fish and birds to mammals and humans, with global socioeconomic negative impacts. Here the authors report and experimentally validate a novel approach for the analysis of the molecular networks that are involved in the recognition of substrates by retroviral proteases. Using multivariate analysis of the sequence-based physiochemical descriptions of 61 retroviral proteases comprising wild-type proteases, natural mutants, and drug-resistant forms of proteases from nine different viral species in relation to their ability to cleave 299 substrates, the authors mapped the physicochemical properties and cross-dependencies of the amino acids of the proteases and their substrates, which revealed a complex molecular interaction network of substrate recognition and cleavage. The approach allowed a detailed analysis of the molecular–chemical mechanisms involved in substrate cleavage by retroviral proteases. PMID:17352531

Substrate recognition by ribonucleoprotein ribonuclease MRP

PubMed Central

Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S.

2011-01-01

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5′ ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed. PMID:21173200

Substrate recognition by ribonucleoprotein ribonuclease MRP.

PubMed

Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S

2011-02-01

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

DOE PAGES

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.; ...

2017-02-23

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less

A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit

PubMed Central

Cundell, Michael J.; Holder, James

2016-01-01

PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis. PMID:27551054

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

PubMed Central

Rashid, Fahad; Harris, Paul D; Zaher, Manal S; Sobhy, Mohamed A; Joudeh, Luay I; Yan, Chunli; Piwonski, Hubert; Tsutakawa, Susan E; Ivanov, Ivaylo; Tainer, John A; Habuchi, Satoshi; Hamdan, Samir M

2017-01-01

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability. DOI: http://dx.doi.org/10.7554/eLife.21884.001 PMID:28230529

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

PubMed Central

Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

2016-01-01

Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and complexed with monoubiquitin or K48-linked diubiquitin were solved, revealing that three neighboring outer helices from the T1 toroid engage two ubiquitins. This binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between deubiquitination and substrate degradation. We find that proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition, but rather in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Importantly, the UBL interactors at T1 and T2 are distinct, assigning substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adjacent receptor sites, T1 and T2. The Rpn1 toroid represents a novel class of binding domains for ubiquitin and UBL proteins. This study thus defines a novel two-site recognition domain intrinsic to the proteasome that uses homologous ubiquitin/UBL-class ligands to assemble substrates, substrate shuttling factors, and a deubiquitinating enzyme in close proximity. A ligand-binding hotspot in the proteasome for assembling substrates and cofactors Schematic (top) and model structure (bottom, left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom, right) Enlarged region of the proteasome to illustrate the Rpn1 T1 and T2 sites bound to a ubiquitin chain (yellow) and deubiquitinating enzyme Ubp6 (green), respectively. PDB 4CR2 and 2B9R were used for this figure. Hundreds of pathways for degradation converge at ubiquitin recognition by proteasome. Here we found that the five known proteasomal ubiquitin receptors are collectively nonessential for ubiquitin recognition, and identified a sixth receptor, Rpn1. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or K48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for K48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus a two-site recognition domain intrinsic to the proteasome uses homologous ubiquitin/UBL-class ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme. PMID:26912900

Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity.

PubMed

Tran, Tam N T; Shelton, Jennifer; Brown, Susan; Durrett, Timothy P

2017-10-01

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol (acetyl-TAG). EaDAcT belongs to a small, plant-specific subfamily of the membrane bound O-acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)-specific marker. By mapping the membrane topology of EaDAcT, we obtained an experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains (TMDs), with both the N- and C-termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl-TAG-producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for EaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of EaDAcT, suggesting that multiple amino acids are important for substrate recognition. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

PubMed Central

Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A.; Yu, Shuai; Hans, Michael; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bosserman, Mary A.; Downey, Theresa; Noinaj, Nicholas

Baeyer–Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C–C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis. Kinetic analyses of selected mutants allowed usmore » to probe the substrate binding pocket of MtmOIV and also to discover the putative NADPH binding site. This is the first substrate-bound structure of MtmOIV providing new insights into substrate recognition and catalysis, which paves the way for the future design of a tailored enzyme for the chemo-enzymatic preparation of novel mithramycin analogues.« less

Structural insights into the cofactor-assisted substrate recognition of yeast methylglyoxal/isovaleraldehyde reductase Gre2.

PubMed

Guo, Peng-Chao; Bao, Zhang-Zhi; Ma, Xiao-Xiao; Xia, Qingyou; Li, Wei-Fang

2014-09-01

Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00Å and NADPH-complexed form at 2.40Å resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

An Aromatic Sensor with Aversion to Damaged Strands Confers Versatility to DNA Repair

PubMed Central

Maillard, Olivier; Solyom, Szilvia; Naegeli, Hanspeter

2007-01-01

It was not known how xeroderma pigmentosum group C (XPC) protein, the primary initiator of global nucleotide excision repair, achieves its outstanding substrate versatility. Here, we analyzed the molecular pathology of a unique Trp690Ser substitution, which is the only reported missense mutation in xeroderma patients mapping to the evolutionary conserved region of XPC protein. The function of this critical residue and neighboring conserved aromatics was tested by site-directed mutagenesis followed by screening for excision activity and DNA binding. This comparison demonstrated that Trp690 and Phe733 drive the preferential recruitment of XPC protein to repair substrates by mediating an exquisite affinity for single-stranded sites. Such a dual deployment of aromatic side chains is the distinctive feature of functional oligonucleotide/oligosaccharide-binding folds and, indeed, sequence homologies with replication protein A and breast cancer susceptibility 2 protein indicate that XPC displays a monomeric variant of this recurrent interaction motif. An aversion to associate with damaged oligonucleotides implies that XPC protein avoids direct contacts with base adducts. These results reveal for the first time, to our knowledge, an entirely inverted mechanism of substrate recognition that relies on the detection of single-stranded configurations in the undamaged complementary sequence of the double helix. PMID:17355181

Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6.

PubMed

Stamos, Jennifer L; Chu, Matthew Ling-Hon; Enos, Michael D; Shah, Niket; Weis, William I

2014-03-18

Glycogen synthase kinase-3 (GSK-3) is a key regulator of many cellular signaling pathways. Unlike most kinases, GSK-3 is controlled by inhibition rather than by specific activation. In the insulin and several other signaling pathways, phosphorylation of a serine present in a conserved sequence near the amino terminus of GSK-3 generates an auto-inhibitory peptide. In contrast, Wnt/β-catenin signal transduction requires phosphorylation of Ser/Pro rich sequences present in the Wnt co-receptors LRP5/6, and these motifs inhibit GSK-3 activity. We present crystal structures of GSK-3 bound to its phosphorylated N-terminus and to two of the phosphorylated LRP6 motifs. A conserved loop unique to GSK-3 undergoes a dramatic conformational change that clamps the bound pseudo-substrate peptides, and reveals the mechanism of primed substrate recognition. The structures rationalize target sequence preferences and suggest avenues for the design of inhibitors selective for a subset of pathways regulated by GSK-3. DOI: http://dx.doi.org/10.7554/eLife.01998.001.

DNA materials: bridging nanotechnology and biotechnology.

PubMed

Yang, Dayong; Hartman, Mark R; Derrien, Thomas L; Hamada, Shogo; An, Duo; Yancey, Kenneth G; Cheng, Ru; Ma, Minglin; Luo, Dan

2014-06-17

CONSPECTUS: In recent decades, DNA has taken on an assortment of diverse roles, not only as the central genetic molecule in biological systems but also as a generic material for nanoscale engineering. DNA possesses many exceptional properties, including its biological function, biocompatibility, molecular recognition ability, and nanoscale controllability. Taking advantage of these unique attributes, a variety of DNA materials have been created with properties derived both from the biological functions and from the structural characteristics of DNA molecules. These novel DNA materials provide a natural bridge between nanotechnology and biotechnology, leading to far-ranging real-world applications. In this Account, we describe our work on the design and construction of DNA materials. Based on the role of DNA in the construction, we categorize DNA materials into two classes: substrate and linker. As a substrate, DNA interfaces with enzymes in biochemical reactions, making use of molecular biology's "enzymatic toolkit". For example, employing DNA as a substrate, we utilized enzymatic ligation to prepare the first bulk hydrogel made entirely of DNA. Using this DNA hydrogel as a structural scaffold, we created a protein-producing DNA hydrogel via linking plasmid DNA onto the hydrogel matrix through enzymatic ligation. Furthermore, to fully make use of the advantages of both DNA materials and polymerase chain reaction (PCR), we prepared thermostable branched DNA that could remain intact even under denaturing conditions, allowing for their use as modular primers for PCR. Moreover, via enzymatic polymerization, we have recently constructed a physical DNA hydrogel with unique internal structure and mechanical properties. As a linker, we have used DNA to interface with other functional moieties, including gold nanoparticles, clay minerals, proteins, and lipids, allowing for hybrid materials with unique properties for desired applications. For example, we recently designed a DNA-protein conjugate as a universal adapter for protein detection. We further demonstrate a diverse assortment of applications for these DNA materials including diagnostics, protein production, controlled drug release systems, the exploration of life evolution, and plasmonics. Although DNA has shown great potential as both substrate and linker in the construction of DNA materials, it is still in the initial stages of becoming a well-established and widely used material. Important challenges include the ease of design and fabrication, scaling-up, and minimizing cost. We envision that DNA materials will continue to bridge the gap between nanotechnology and biotechnology and will ultimately be employed for many real-world applications.

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

PubMed

Ngo, Tri Duc; Van Le, Binh; Subramani, Vinod Kumar; Thi Nguyen, Chi My; Lee, Hyun Sook; Cho, Yona; Kim, Kyeong Kyu; Hwang, Hye-Yeon

2015-05-22

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode. Copyright © 2015 Elsevier Inc. All rights reserved.

Phosphine-substrate recognition through the C-H...O hydrogen bond: application to the asymmetric Pauson-Khand reaction.

PubMed

Solà, Jordi; Riera, Antoni; Verdaguer, Xavier; Maestro, Miguel A

2005-10-05

A unique methine moiety attached to three heteroatoms (O, P, S) and contained in the PuPHOS and CamPHOS ligands serves as a strong hydrogen-bond donor. Nonclassical hydrogen bonding of this methine with an amido-carbonyl acceptor provides a completely diastereoselective ligand exchange process between an alkyne dicobalthexacarbonyl complex and a phosphine ligand. This weak contact has been studied by means of X-ray analysis, 1H NMR, and quantum mechanical calculations and revealed that the present interaction falls in the range of strong C-H...O=C bonds. The hydrogen-bond bias obtained in the ligand exchange process has been exploited in the asymmetric intermolecular Pauson-Khand reaction to yield the corresponding cyclization adducts in up to 94% ee.

SCFβ-TrCP ubiquitin ligase-mediated processing of NF-κB p105 requires phosphorylation of its C-terminus by IκB kinase

PubMed Central

Orian, Amir; Gonen, Hedva; Bercovich, Beatrice; Fajerman, Ifat; Eytan, Esther; Israël, Alain; Mercurio, Frank; Iwai, Kazuhiro; Schwartz, Alan L.; Ciechanover, Aaron

2000-01-01

Processing of the p105 precursor to form the active subunit p50 of the NF-κB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine-rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Here we demonstrate that following IκB kinase (IκK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918–934) serves as a recognition motif for the SCFβ-TrCP ubiquitin ligase. Expression of IκKβ dramatically increases processing of wild-type p105, but not of p105-Δ918–934. Dominant-negative β-TrCP inhibits IκK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-Δ918–934 associate physically following phosphorylation. In vitro, SCFβ-TrCP specifically conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IκBs, β-catenin and human immunodeficiency virus type 1 Vpu. Since p105-Δ918–934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. PMID:10835356

Molecular evolution of the CYP2D subfamily in primates: purifying selection on substrate recognition sites without the frequent or long-tract gene conversion.

PubMed

Yasukochi, Yoshiki; Satta, Yoko

2015-03-25

The human cytochrome P450 (CYP) 2D6 gene is a member of the CYP2D gene subfamily, along with the CYP2D7P and CYP2D8P pseudogenes. Although the CYP2D6 enzyme has been studied extensively because of its clinical importance, the evolution of the CYP2D subfamily has not yet been fully understood. Therefore, the goal of this study was to reveal the evolutionary process of the human drug metabolic system. Here, we investigate molecular evolution of the CYP2D subfamily in primates by comparing 14 CYP2D sequences from humans to New World monkey genomes. Window analysis and statistical tests revealed that entire genomic sequences of paralogous genes were extensively homogenized by gene conversion during molecular evolution of CYP2D genes in primates. A neighbor-joining tree based on genomic sequences at the nonsubstrate recognition sites showed that CYP2D6 and CYP2D8 genes were clustered together due to gene conversion. In contrast, a phylogenetic tree using amino acid sequences at substrate recognition sites did not cluster the CYP2D6 and CYP2D8 genes, suggesting that the functional constraint on substrate specificity is one of the causes for purifying selection at the substrate recognition sites. Our results suggest that the CYP2D gene subfamily in primates has evolved to maintain the regioselectivity for a substrate hydroxylation activity between individual enzymes, even though extensive gene conversion has occurred across CYP2D coding sequences. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Molecular Evolution of the CYP2D Subfamily in Primates: Purifying Selection on Substrate Recognition Sites without the Frequent or Long-Tract Gene Conversion

PubMed Central

Yasukochi, Yoshiki; Satta, Yoko

2015-01-01

The human cytochrome P450 (CYP) 2D6 gene is a member of the CYP2D gene subfamily, along with the CYP2D7P and CYP2D8P pseudogenes. Although the CYP2D6 enzyme has been studied extensively because of its clinical importance, the evolution of the CYP2D subfamily has not yet been fully understood. Therefore, the goal of this study was to reveal the evolutionary process of the human drug metabolic system. Here, we investigate molecular evolution of the CYP2D subfamily in primates by comparing 14 CYP2D sequences from humans to New World monkey genomes. Window analysis and statistical tests revealed that entire genomic sequences of paralogous genes were extensively homogenized by gene conversion during molecular evolution of CYP2D genes in primates. A neighbor-joining tree based on genomic sequences at the nonsubstrate recognition sites showed that CYP2D6 and CYP2D8 genes were clustered together due to gene conversion. In contrast, a phylogenetic tree using amino acid sequences at substrate recognition sites did not cluster the CYP2D6 and CYP2D8 genes, suggesting that the functional constraint on substrate specificity is one of the causes for purifying selection at the substrate recognition sites. Our results suggest that the CYP2D gene subfamily in primates has evolved to maintain the regioselectivity for a substrate hydroxylation activity between individual enzymes, even though extensive gene conversion has occurred across CYP2D coding sequences. PMID:25808902

Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating Activity of SARS-CoV Papain-Like Protease

PubMed Central

Ratia, Kiira; Kilianski, Andrew; Baez-Santos, Yahira M.; Baker, Susan C.; Mesecar, Andrew

2014-01-01

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response. PMID:24854014

Infrared sensing of non-observable human biometrics

NASA Astrophysics Data System (ADS)

Willmore, Michael R.

2005-05-01

Interest and growth of biometric recognition technologies surged after 9/11. Once a technology mainly used for identity verification in law enforcement, biometrics are now being considered as a secure means of providing identity assurance in security related applications. Biometric recognition in law enforcement must, by necessity, use attributes of human uniqueness that are both observable and vulnerable to compromise. Privacy and protection of an individual's identity is not assured during criminal activity. However, a security system must rely on identity assurance for access control to physical or logical spaces while not being vulnerable to compromise and protecting the privacy of an individual. The solution resides in the use of non-observable attributes of human uniqueness to perform the biometric recognition process. This discussion will begin by presenting some key perspectives about biometric recognition and the characteristic differences between observable and non-observable biometric attributes. An introduction to the design, development, and testing of the Thermo-ID system will follow. The Thermo-ID system is an emerging biometric recognition technology that uses non-observable patterns of infrared energy naturally emanating from within the human body. As with all biometric systems, the infrared patterns recorded and compared within the Thermo-ID system are unique and individually distinguishable permitting a link to be confirmed between an individual and a claimed or previously established identity. The non-observable characteristics of infrared patterns of human uniqueness insure both the privacy and protection of an individual using this type of biometric recognition system.

Triggered optical biosensor

DOEpatents

Song, Xuedong; Swanson, Basil I.

2001-10-02

An optical biosensor is provided for the detection of a multivalent target biomolecule, the biosensor including a substrate having a bilayer membrane thereon, a recognition molecule situated at the surface, the recognition molecule capable of binding with the multivalent target biomolecule, the recognition molecule further characterized as including a fluorescence label thereon and as being movable at the surface and a device for measuring a fluorescence change in response to binding between the recognition molecule and the multivalent target biomolecule.

[A new mechanism of ubiquitin-dependent proteolytic pathway--polyubiquitin chain recognition and proteasomal targeting].

PubMed

Kawahara, Hiroyuki; Yokosawa, Hideyoshi

2008-01-01

The RPN10 subunit of 26S proteasome and several UBA domain proteins can bind to the polyubiquitin chain and play a role as ubiquitin receptors of the 26S proteasome. Although it was thought that substrate recognition is an essential step in the proteasome-mediated protein degradation, deletion of rpn10 genes in yeast does not influence the viability of cells but instead causes only a mild phenotype, suggesting that the above ubiquitin receptors are redundantly involved in substrate delivery to the proteasome. However, their functional difference is still enigmatic. In this review, we summarize recent advances in polyubiquitin chain recognition/delivery system and provide potential applications to modulate this system as a probable target for drug development.

Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

PubMed Central

Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

2011-01-01

HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

DOE PAGES

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

2017-07-07

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Enzyme-substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases.

PubMed

O'Connor, Hazel F; Huibregtse, Jon M

2017-09-01

Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3-substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein-protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.

Substrate-based inhibitors exhibiting excellent protective and therapeutic effects against Botulinum Neurotoxin A intoxication.

PubMed

Guo, Jiubiao; Wang, Jinglin; Gao, Shan; Ji, Bin; Waichi Chan, Edward; Chen, Sheng

2015-11-20

Potent inhibitors to reverse Botulinum neurotoxins (BoNTs) activity in neuronal cells are currently not available. A better understanding of the substrate recognition mechanism of BoNTs enabled us to design a novel class of peptide inhibitors which were derivatives of the BoNT/A substrate, SNAP25. Through a combination of in vitro, cellular based, and in vivo mouse assays, several potent inhibitors of approximately one nanomolar inhibitory strength both in vitro and in vivo have been identified. These compounds represent the first set of inhibitors that exhibited full protection against BoNT/A intoxication in mice model with undetectable toxicity. Our findings validated the hypothesis that a peptide inhibitor targeting the two BoNT structural regions which were responsible for substrate recognition and cleavage respectively could exhibit excellent inhibitory effect, thereby providing insight on future development of more potent inhibitors against BoNTs.

Cortical Networks for Visual Self-Recognition

NASA Astrophysics Data System (ADS)

Sugiura, Motoaki

This paper briefly reviews recent developments regarding the brain mechanisms of visual self-recognition. A special cognitive mechanism for visual self-recognition has been postulated based on behavioral and neuropsychological evidence, but its neural substrate remains controversial. Recent functional imaging studies suggest that multiple cortical mechanisms play self-specific roles during visual self-recognition, reconciling the existing controversy. Respective roles for the left occipitotemporal, right parietal, and frontal cortices in symbolic, visuospatial, and conceptual aspects of self-representation have been proposed.

Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant path...

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3-1,4-Glucans through Distinct Mechanisms.

PubMed

Venditto, Immacolata; Najmudin, Shabir; Luís, Ana S; Ferreira, Luís M A; Sakka, Kazuo; Knox, J Paul; Gilbert, Harry J; Fontes, Carlos M G A

2015-04-24

Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with β-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a β-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to β-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to β-1,3-1,4-glucans while substantially decreasing the affinity for decorated β-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified β-1,3-1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The B1 Protein Guides the Biosynthesis of a Lasso Peptide

NASA Astrophysics Data System (ADS)

Zhu, Shaozhou; Fage, Christopher D.; Hegemann, Julian D.; Mielcarek, Andreas; Yan, Dushan; Linne, Uwe; Marahiel, Mohamed A.

2016-10-01

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold that endows them with extraordinary stability and biologically relevant activity. However, the biosynthetic mechanism of these fascinating molecules remains largely speculative. Generally, two enzymes (B for processing and C for cyclization) are required to assemble the unusual knot-like structure. Several subsets of lasso peptide gene clusters feature a “split” B protein on separate open reading frames (B1 and B2), suggesting distinct functions for the B protein in lasso peptide biosynthesis. Herein, we provide new insights into the role of the RiPP recognition element (RRE) PadeB1, characterizing its capacity to bind the paeninodin leader peptide and deliver its peptide substrate to PadeB2 for processing.

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control.

PubMed

Satoh, Tadashi; Toshimori, Takayasu; Noda, Masanori; Uchiyama, Susumu; Kato, Koichi

2016-11-01

The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme. © 2016 The Protein Society.

Top surface blade residues and the central channel water molecules are conserved in every repeat of the integrin-like β-propeller structures.

PubMed

Denesyuk, Alexander; Denessiouk, Konstantin; Johnson, Mark S

2018-02-01

An integrin-like β-propeller domain contains seven repeats of a four-stranded antiparallel β-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type β-propeller. Here, we show unique structural links that join different blades of the β-propeller structure, which together with the structural motif for a single blade are repeated in a β-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition. We compare functional top face diagrams of the integrin-type β-propeller domain and two non-integrin type β-propeller domains of virginiamycin B lyase and WD Repeat-Containing Protein 5. Copyright © 2017 Elsevier Inc. All rights reserved.

Screen-printed nanoparticles as anti-counterfeiting tags

NASA Astrophysics Data System (ADS)

Campos-Cuerva, Carlos; Zieba, Maciej; Sebastian, Victor; Martínez, Gema; Sese, Javier; Irusta, Silvia; Contamina, Vicente; Arruebo, Manuel; Santamaria, Jesus

2016-03-01

Metallic nanoparticles with different physical properties have been screen printed as authentication tags on different types of paper. Gold and silver nanoparticles show unique optical signatures, including sharp emission bandwidths and long lifetimes of the printed label, even under accelerated weathering conditions. Magnetic nanoparticles show distinct physical signals that depend on the size of the nanoparticle itself. They were also screen printed on different substrates and their magnetic signals read out using a magnetic pattern recognition sensor and a vibrating sample magnetometer. The novelty of our work lies in the demonstration that the combination of nanomaterials with optical and magnetic properties on the same printed support is possible, and the resulting combined signals can be used to obtain a user-configurable label, providing a high degree of security in anti-counterfeiting applications using simple commercially-available sensors.

Screen-printed nanoparticles as anti-counterfeiting tags.

PubMed

Campos-Cuerva, Carlos; Zieba, Maciej; Sebastian, Victor; Martínez, Gema; Sese, Javier; Irusta, Silvia; Contamina, Vicente; Arruebo, Manuel; Santamaria, Jesus

2016-03-04

Metallic nanoparticles with different physical properties have been screen printed as authentication tags on different types of paper. Gold and silver nanoparticles show unique optical signatures, including sharp emission bandwidths and long lifetimes of the printed label, even under accelerated weathering conditions. Magnetic nanoparticles show distinct physical signals that depend on the size of the nanoparticle itself. They were also screen printed on different substrates and their magnetic signals read out using a magnetic pattern recognition sensor and a vibrating sample magnetometer. The novelty of our work lies in the demonstration that the combination of nanomaterials with optical and magnetic properties on the same printed support is possible, and the resulting combined signals can be used to obtain a user-configurable label, providing a high degree of security in anti-counterfeiting applications using simple commercially-available sensors.

Molecular Basis of Substrate Recognition and Degradation by Human Presequence Protease

PubMed Central

King, John V.; Liang, Wenguang G.; Scherpelz, Kathryn P.; Schilling, Alexander B.; Meredith, Stephen C.; Tang, Wei-Jen

2014-01-01

Summary Human Presequence Protease (hPreP) is an M16 metalloprotease localized in mitochondria. There, hPreP facilitates proteostasis by utilizing a ∼13,300Å3 catalytic chamber to degrade a diverse array of potentially toxic peptides, including mitochondrial presequences and amyloid-β (Aβ), the latter of which contributes to Alzheimer's disease pathogenesis. Here we report crystal structures for hPreP alone and in complex with Aβ, which show that hPreP uses size-exclusion and charge complementation for substrate recognition. These structures also reveal hPreP-specific features that permit a diverse array of peptides, with distinct distributions of charged and hydrophobic residues, to be specifically captured, cleaved, and their amyloidogenic features destroyed. SAXS analysis demonstrates that hPreP in solution exists in dynamic equilibrium between closed and open states, with the former being preferred. Furthermore, Aβ binding induces the closed state and hPreP dimerization. Together, these data reveal the molecular basis for flexible yet specific substrate recognition and degradation by hPreP. PMID:24931469

Recognition of Acyl Carrier Proteins by Ketoreductases in Assembly Line Polyketide Synthases

PubMed Central

Ostrowski, Matthew P.; Cane, David E.; Khosla, Chaitan

2016-01-01

Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed a ca. 10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed a ca. 10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules. PMID:27118242

Simulation studies of substrate recognition by the exocellulase CelF from Clostridium cellulolyticum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Mo; Himmel, Michael E.; Wilson, David B.

Molecular dynamics (MD) simulations were used to study substrate recognition by the family 48 exocellulase CelF from Clostridium cellulolyticum. It was hypothesized that residues around the entrance of the active site tunnel of this enzyme might serve to recognize and bind the substrate through an affinity for the cellulose monomer repeat unit, ..beta..-d-glucopyranose. Simulations were conducted of the catalytic domain of this enzyme surrounded by a concentrated solution of ..beta..-d-glucopyranose, and the full three-dimensional probability distribution for finding sugar molecules adjacent to the enzyme was calculated from the trajectory. A significant probability of finding the sugar stacked against the planarmore » faces of Trp 310 and Trp 312 at the entrance of the active site tunnel was observed.« less

Shared Neural Substrates of Emotionally Enhanced Perceptual and Mnemonic Vividness

PubMed Central

Todd, Rebecca M.; Schmitz, Taylor W.; Susskind, Josh; Anderson, Adam K.

2013-01-01

It is well-known that emotionally salient events are remembered more vividly than mundane ones. Our recent research has demonstrated that such memory vividness (Mviv) is due in part to the subjective experience of emotional events as more perceptually vivid, an effect we call emotionally enhanced vividness (EEV). The present study built on previously reported research in which fMRI data were collected while participants rated relative levels of visual noise overlaid on emotionally salient and neutral images. Ratings of greater EEV were associated with greater activation in the amygdala and visual cortex. In the present study, we measured BOLD activation that predicted recognition Mviv for these same images 1 week later. Results showed that, after controlling for differences between scenes in low-level objective features, hippocampus activation uniquely predicted subsequent Mviv. In contrast, amygdala and visual cortex regions that were sensitive to EEV were also modulated by subsequent ratings of Mviv. These findings suggest shared neural substrates for the influence of emotional salience on perceptual and mnemonic vividness, with amygdala and visual cortex activation at encoding contributing to the experience of both perception and subsequent memory. PMID:23653601

Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP).

PubMed

Bainbridge, Travis W; Dunshee, Diana Ronai; Kljavin, Noelyn M; Skelton, Nicholas J; Sonoda, Junichiro; Ernst, James A

2017-10-02

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

Our “energy-Ca2+ signaling deficits” hypothesis and its explanatory potential for key features of Alzheimer’s disease

PubMed Central

Chen, Ming; Nguyen, Huey T.

2014-01-01

Sporadic Alzheimer’s disease (sAD) has not been explained by any current theories, so new hypotheses are urgently needed. We proposed that “energy and Ca2+ signaling deficits” are perhaps the earliest modifiable defects in brain aging underlying memory decline and tau deposits (by means of inactivating Ca2+-dependent protease calpain). Consistent with this hypothesis, we now notice that at least eight other known calpain substrates have also been reported to accumulate in aging and AD. Thus, protein accumulation or aggregation is not a “pathogenic” event, but occurs naturally and selectively to a peculiar family of proteins, and is best explained by calpain inactivation. Why are only calpain substrates accumulated and how can they stay for decades in the brain without being attacked by many other non-specific proteases there? We believe that these long-lasting puzzles can be explained by calpain’s unique properties, especially its unusual specificity and exclusivity in substrate recognition, which can protect the substrates from other proteases’ attacks after calpain inactivation. Interestingly, our model, in essence, may also explain tau phosphorylation and the formation of amyloid plaques. Our studies suggest that α-secretase is an energy-/Ca2+-dual dependent protease and is also the primary determinant for Aβ levels. Therefore, β- and γ-secretases can only play secondary roles and, by biological laws, they are unlikely to be “positively identified”. This study thus raises serious questions for policymakers and researchers and these questions may help explain why sAD can remain an enigma today. PMID:25489296

Structure of a [NiFe] hydrogenase maturation protease HycI provides insights into its substrate selectivity.

PubMed

Kwon, Sunghark; Nishitani, Yuichi; Hirao, Yoshinori; Kanai, Tamotsu; Atomi, Haruyuki; Miki, Kunio

2018-04-15

The immature large subunit of [NiFe] hydrogenases undergoes C-terminal cleavage by a specific protease in the final step of the post-translational process before assembly with other subunits. It has been reported that the [NiFe] hydrogenase maturation protease HycI from Thermococcus kodakarensis (TkHycI) has the catalytic ability to target the membrane-bound hydrogenase large subunit MbhL from T. kodakarensis. However, the detailed mechanism of its substrate recognition remains elusive. We determined the crystal structure of TkHycI at 1.59 Å resolution to clarify how TkHycI recognizes its own substrate MbhL. Although the overall structure of TkHycI is similar to that of its homologous protease TkHybD, TkHycI adopts a larger loop than TkHybD, thereby creating a broad and deep cleft. We analyzed the structural properties of the TkHycI cleft probably involved in its substrate recognition. Our findings provide novel and profound insights into the substrate selectivity of TkHycI. Copyright © 2018 Elsevier Inc. All rights reserved.

The structural neuroanatomy of music emotion recognition: Evidence from frontotemporal lobar degeneration

PubMed Central

Omar, Rohani; Henley, Susie M.D.; Bartlett, Jonathan W.; Hailstone, Julia C.; Gordon, Elizabeth; Sauter, Disa A.; Frost, Chris; Scott, Sophie K.; Warren, Jason D.

2011-01-01

Despite growing clinical and neurobiological interest in the brain mechanisms that process emotion in music, these mechanisms remain incompletely understood. Patients with frontotemporal lobar degeneration (FTLD) frequently exhibit clinical syndromes that illustrate the effects of breakdown in emotional and social functioning. Here we investigated the neuroanatomical substrate for recognition of musical emotion in a cohort of 26 patients with FTLD (16 with behavioural variant frontotemporal dementia, bvFTD, 10 with semantic dementia, SemD) using voxel-based morphometry. On neuropsychological evaluation, patients with FTLD showed deficient recognition of canonical emotions (happiness, sadness, anger and fear) from music as well as faces and voices compared with healthy control subjects. Impaired recognition of emotions from music was specifically associated with grey matter loss in a distributed cerebral network including insula, orbitofrontal cortex, anterior cingulate and medial prefrontal cortex, anterior temporal and more posterior temporal and parietal cortices, amygdala and the subcortical mesolimbic system. This network constitutes an essential brain substrate for recognition of musical emotion that overlaps with brain regions previously implicated in coding emotional value, behavioural context, conceptual knowledge and theory of mind. Musical emotion recognition may probe the interface of these processes, delineating a profile of brain damage that is essential for the abstraction of complex social emotions. PMID:21385617

The structural neuroanatomy of music emotion recognition: evidence from frontotemporal lobar degeneration.

PubMed

Omar, Rohani; Henley, Susie M D; Bartlett, Jonathan W; Hailstone, Julia C; Gordon, Elizabeth; Sauter, Disa A; Frost, Chris; Scott, Sophie K; Warren, Jason D

2011-06-01

Despite growing clinical and neurobiological interest in the brain mechanisms that process emotion in music, these mechanisms remain incompletely understood. Patients with frontotemporal lobar degeneration (FTLD) frequently exhibit clinical syndromes that illustrate the effects of breakdown in emotional and social functioning. Here we investigated the neuroanatomical substrate for recognition of musical emotion in a cohort of 26 patients with FTLD (16 with behavioural variant frontotemporal dementia, bvFTD, 10 with semantic dementia, SemD) using voxel-based morphometry. On neuropsychological evaluation, patients with FTLD showed deficient recognition of canonical emotions (happiness, sadness, anger and fear) from music as well as faces and voices compared with healthy control subjects. Impaired recognition of emotions from music was specifically associated with grey matter loss in a distributed cerebral network including insula, orbitofrontal cortex, anterior cingulate and medial prefrontal cortex, anterior temporal and more posterior temporal and parietal cortices, amygdala and the subcortical mesolimbic system. This network constitutes an essential brain substrate for recognition of musical emotion that overlaps with brain regions previously implicated in coding emotional value, behavioural context, conceptual knowledge and theory of mind. Musical emotion recognition may probe the interface of these processes, delineating a profile of brain damage that is essential for the abstraction of complex social emotions. Copyright © 2011 Elsevier Inc. All rights reserved.

The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.

Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. In this paper, we present the first, to ourmore » knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge–dipole and CH–π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Finally and hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.« less

The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

DOE PAGES

Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.; ...

2015-10-28

Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. In this paper, we present the first, to ourmore » knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge–dipole and CH–π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Finally and hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.« less

An Evaluation of PC-Based Optical Character Recognition Systems.

ERIC Educational Resources Information Center

Schreier, E. M.; Uslan, M. M.

1991-01-01

The review examines six personal computer-based optical character recognition (OCR) systems designed for use by blind and visually impaired people. Considered are OCR components and terms, documentation, scanning and reading, command structure, conversion, unique features, accuracy of recognition, scanning time, speed, and cost. (DB)

Plant recognition of Bradyrhizobium japonicum nod factors. Final report, September 15, 1992--March 14, 1997

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stacey, G.

1998-01-01

This grant had three objectives: (1) isolate and identify the unique nod factor metabolites made by different wild-type B. japonicum strains; (2) investigate the biological activity of these unique nod factors, especially as it relates to host range; and (3) initiate studies to define the mechanism of plant recognition of the nod factors. This report summarizes the results of this research.

Fractionating the Neural Substrates of Incidental Recognition Memory

ERIC Educational Resources Information Center

Greene, Ciara M.; Vidaki, Kleio; Soto, David

2015-01-01

Familiar stimuli are typically accompanied by decreases in neural response relative to the presentation of novel items, but these studies often include explicit instructions to discriminate old and new items; this creates difficulties in partialling out the contribution of top-down intentional orientation to the items based on recognition goals.…

Recognition Imaging with a DNA Aptamer

PubMed Central

Lin, Liyun; Wang, Hongda; Liu, Yan; Yan, Hao; Lindsay, Stuart

2006-01-01

We have used a DNA-aptamer tethered to an atomic force microscope probe to carry out recognition imaging of IgE molecules attached to a mica substrate. The recognition was efficient (∼90%) and specific, being blocked by injection of IgE molecules in solution, and not being interfered with by high concentrations of a second protein. The signal/noise ratio of the recognition signal was better than that obtained with antibodies, despite the fact that the average force required to break the aptamer-protein bonds was somewhat smaller. PMID:16513776

Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

PubMed

Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

2018-06-16

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

Retinoic acid biosynthesis catalyzed by retinal dehydrogenases relies on a rate-limiting conformational transition associated with substrate recognition

PubMed Central

Bchini, Raphaël; Vasiliou, Vasilis; Branlant, Guy; Talfournier, François; Rahuel-Clermont, Sophie

2012-01-01

Retinoic acid (RA), a metabolite of vitamin A, exerts pleiotropic effects throughout life in vertebrate organisms. Thus, RA action must be tightly regulated through the coordinated action of biosynthetic and degradating enzymes. The last step of retinoic acid biosynthesis is irreversibly catalyzed by the NAD-dependent retinal dehydrogenases (RALDH), which are members of the aldehyde dehydrogenase (ALDH) superfamily. Low intracellular retinal concentrations imply efficient substrate molecular recognition to ensure high affinity and specificity of RALDHs for retinal. This study addresses the molecular basis of retinal recognition in human ALDH1A1 (or RALDH1) and rat ALDH1A2 (or RALDH2), through the comparison of the catalytic behavior of retinal analogs and use of the fluorescence properties of retinol. We show that, in contrast to long chain unsaturated substrates, the rate-limiting step of retinal oxidation by RALDHs is associated with acylation. Use of the fluorescence resonance energy transfer upon retinol interaction with RALDHs provides evidence that retinal recognition occurs in two steps: binding into the substrate access channel, and a slower structural reorganization with a rate constant of the same magnitude as the kcat for retinal oxidation: 0.18 vs. 0.07 s−1 and 0.25 vs. 0.1 s−1 for ALDH1A1 and ALDH1A2, respectively. This suggests that the conformational transition of the RALDH-retinal complex significantly contributes to the rate-limiting step that controls the kinetics of retinal oxidation, as a prerequisite for the formation of a catalytically competent Michaelis complex. This conclusion is consistent with the general notion that structural flexibility within the active site of ALDH enzymes has been shown to be an integral component of catalysis. PMID:23220587

Fourier transform magnitudes are unique pattern recognition templates.

PubMed

Gardenier, P H; McCallum, B C; Bates, R H

1986-01-01

Fourier transform magnitudes are commonly used in the generation of templates in pattern recognition applications. We report on recent advances in Fourier phase retrieval which are relevant to pattern recognition. We emphasise in particular that the intrinsic form of a finite, positive image is, in general, uniquely related to the magnitude of its Fourier transform. We state conditions under which the Fourier phase can be reconstructed from samples of the Fourier magnitude, and describe a method of achieving this. Computational examples of restoration of Fourier phase (and hence, by Fourier transformation, the intrinsic form of the image) from samples of the Fourier magnitude are also presented.

Contributions of a unique β-clamp to substrate recognition illuminates the molecular basis of exolysis in ferulic acid esterases.

PubMed

Gruninger, Robert J; Cote, Chris; McAllister, Tim A; Abbott, D Wade

2016-04-01

Lignocellulosic biomass is a promising renewable resource; however, deconstruction of this material is still the rate-limiting step. Major obstacles in the biocatalytic turnover of lignocellulose are ester-linked decorations that prevent access to primary structural polysaccharides. Enzymes targeting these esters represent promising biotools for increasing bioconversion efficiency. Ruminant livestock are unique in their ability to degrade lignocellulose through the action of their gut microbiome. The anaerobic fungi (phylum Neocallimastigomycota) are key members of this ecosystem that express a large repertoire of carbohydrate-active enzymes (CAZymes) with little sequence identity with characterized CAZymes [Lombard, Golaconda, Drula, Coutinho and Henrissat (2014) Nucleic Acids Res. 42: , D490-D495]. We have identified a carbohydrate esterase family 1 (CE1) ferulic acid esterase (FAE) belonging to Anaeromyces mucronatus(AmCE1/Fae1a), and determined its X-ray structure in both the presence [1.55 Å (1 Å=0.1 nm)] and absence (1.60 Å) of ferulic acid. AmCE1 adopts an α/β-hydrolase fold that is structurally conserved with bacterial FAEs, and possesses a unique loop, termed the β-clamp, that encloses the ligand. Isothermal titration calorimetry reveals that substrate binding is driven by enthalpic contributions, which overcomes a large entropic penalty. A comparative analysis of AmCE1 with related enzymes has uncovered the apparent structural basis for differential FAE activities targeting cross-linking ferulic acid conjugates compared with terminal decorations. Based on comparisons to structurally characterized FAEs, we propose that the β-clamp may define the structural basis of exolytic activities in FAEs. This provides a structure-based tool for predicting exolysis and endolysis in CE1. These insights hold promise for rationally identifying enzymes tailored for bioconversion of biomass with variations in cell wall composition. © 2016 Authors; published by Portland Press Limited.

Structure and Function of the First Full-Length Murein Peptide Ligase (Mpl) Cell Wall Recycling Protein

PubMed Central

Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L.; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W.; Klock, Heath E.; Miller, Mitchell D.; Godzik, Adam; Lesley, Scott A.; Deacon, Ashley M.; Mengin-Lecreulx, Dominique; Wilson, Ian A.

2011-01-01

Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships. PMID:21445265

Structure and function of the first full-length murein peptide ligase (Mpl) cell wall recycling protein.

PubMed

Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W; Klock, Heath E; Miller, Mitchell D; Godzik, Adam; Lesley, Scott A; Deacon, Ashley M; Mengin-Lecreulx, Dominique; Wilson, Ian A

2011-03-18

Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In gram-negative bacteria, ∼30-60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships.

Structure of an XPF endonuclease with and without DNA suggests a model for substrate recognition

PubMed Central

Newman, Matthew; Murray-Rust, Judith; Lally, John; Rudolf, Jana; Fadden, Andrew; Knowles, Philip P; White, Malcolm F; McDonald, Neil Q

2005-01-01

The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)2 domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3′ flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes. PMID:15719018

United States Homeland Security and National Biometric Identification

DTIC Science & Technology

2002-04-09

security number. Biometrics is the use of unique individual traits such as fingerprints, iris eye patterns, voice recognition, and facial recognition to...technology to control access onto their military bases using a Defense Manpower Management Command developed software application. FACIAL Facial recognition systems...installed facial recognition systems in conjunction with a series of 200 cameras to fight street crime and identify terrorists. The cameras, which are

Saccharide sensing molecules having enhanced fluorescent properties

DOEpatents

Satcher Jr., Joe H.; Lane, Stephen M.; Darrow, Christopher B.; Cary, Douglas R.; Tran, Joe Anh

2004-01-06

The present invention provides formulae for fluorescent compounds that have a number of properties which make them uniquely suited for use in sensors of analytes such as saccharides. The advantageous fluorescent properties include favorable excitation wavelengths, emission wavelengths, fluorescence lifetimes, and photostability. Additional advantageous properties include enhanced aqueous solubility, as well as temperature and pH sensitivity. The compound comprises an aryl or a substituted phenyl botonic acid that acts as a substrate recognition component, a fluorescence switch component, and a fluorophore. Fluorescent compounds are described that are excited at wavelengths greater than 400 nm and emit at wavelengths greater than 450 nm, which is advantageous for optical transmission through skin. The fluorophore is typically selected from transition metal-ligand complexes and thiazine, oxazine, oxazone, or oxazine-one as well as anthracene compounds. The fluorescent compound can be immobilized in a glucose permeable biocompatible polymer matrix that is implantable below the skin.

Patterned Colloidal Photonic Crystals.

PubMed

Hou, Jue; Li, Mingzhu; Song, Yanlin

2018-03-01

Colloidal photonic crystals (PCs) have been well developed because they are easy to prepare, cost-effective, and versatile with regards to modification and functionalization. Patterned colloidal PCs contribute a novel approach to constructing high-performance PC devices with unique structures and specific functions. In this review, an overview of the strategies for fabricating patterned colloidal PCs, including patterned substrate-induced assembly, inkjet printing, and selective immobilization and modification, is presented. The advantages of patterned PC devices are also discussed in detail, for example, improved detection sensitivity and response speed of the sensors, control over the flow direction and wicking rate of microfluidic channels, recognition of cross-reactive molecules through an array-patterned microchip, fabrication of display devices with tunable patterns, well-arranged RGB units, and wide viewing-angles, and the ability to construct anti-counterfeiting devices with different security strategies. Finally, the perspective of future developments and challenges is presented. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis *

PubMed Central

Schiebel, Johannes; Kapilashrami, Kanishk; Fekete, Agnes; Bommineni, Gopal R.; Schaefer, Christin M.; Mueller, Martin J.; Tonge, Peter J.; Kisker, Caroline

2013-01-01

The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60–90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C38–42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors. PMID:24108128

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases*

PubMed Central

Klaus, Maja; Ostrowski, Matthew P.; Austerjost, Jonas; Robbins, Thomas; Lowry, Brian; Cane, David E.; Khosla, Chaitan

2016-01-01

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs. PMID:27246853

Age differences in right-wing authoritarianism and their relation to emotion recognition.

PubMed

Ruffman, Ted; Wilson, Marc; Henry, Julie D; Dawson, Abigail; Chen, Yan; Kladnitski, Natalie; Myftari, Ella; Murray, Janice; Halberstadt, Jamin; Hunter, John A

2016-03-01

This study examined the correlates of right-wing authoritarianism (RWA) in older adults. Participants were given tasks measuring emotion recognition, executive functions and fluid IQ and questionnaires measuring RWA, perceived threat and social dominance orientation. Study 1 established higher age-related RWA across the age span in more than 2,600 New Zealanders. Studies 2 to 4 found that threat, education, social dominance and age all predicted unique variance in older adults' RWA, but the most consistent predictor was emotion recognition, predicting unique variance in older adults' RWA independent of all other variables. We argue that older adults' worse emotion recognition is associated with a more general change in social judgment. Expression of extreme attitudes (right- or left-wing) has the potential to antagonize others, but worse emotion recognition means that subtle signals will not be perceived, making the expression of extreme attitudes more likely. Our findings are consistent with other studies showing that worsening emotion recognition underlies age-related declines in verbosity, understanding of social gaffes, and ability to detect lies. Such results indicate that emotion recognition is a core social insight linked to many aspects of social cognition. (c) 2016 APA, all rights reserved).

The plastid and mitochondrial peptidase network in Arabidopsis thaliana: a foundation for testing genetic interactions and functions in organellar proteostasis

USDA-ARS?s Scientific Manuscript database

Plant plastids and mitochondria have dynamic proteomes. To maintain their protein homeostasis, a proteostasis network containing protein chaperones, peptidases and their substrate recognition factors exists, but many peptidases, their functional connections and substrates are poorly characterized. T...

An enhanced feature set for pattern recognition based contrast enhancement of contact-less captured latent fingerprints in digitized crime scene forensics

NASA Astrophysics Data System (ADS)

Hildebrandt, Mario; Kiltz, Stefan; Dittmann, Jana; Vielhauer, Claus

2014-02-01

In crime scene forensics latent fingerprints are found on various substrates. Nowadays primarily physical or chemical preprocessing techniques are applied for enhancing the visibility of the fingerprint trace. In order to avoid altering the trace it has been shown that contact-less sensors offer a non-destructive acquisition approach. Here, the exploitation of fingerprint or substrate properties and the utilization of signal processing techniques are an essential requirement to enhance the fingerprint visibility. However, especially the optimal sensory is often substrate-dependent. An enhanced generic pattern recognition based contrast enhancement approach for scans of a chromatic white light sensor is introduced in Hildebrandt et al.1 using statistical, structural and Benford's law2 features for blocks of 50 micron. This approach achieves very good results for latent fingerprints on cooperative, non-textured, smooth substrates. However, on textured and structured substrates the error rates are very high and the approach thus unsuitable for forensic use cases. We propose the extension of the feature set with semantic features derived from known Gabor filter based exemplar fingerprint enhancement techniques by suggesting an Epsilon-neighborhood of each block in order to achieve an improved accuracy (called fingerprint ridge orientation semantics). Furthermore, we use rotation invariant Hu moments as an extension of the structural features and two additional preprocessing methods (separate X- and Y Sobel operators). This results in a 408-dimensional feature space. In our experiments we investigate and report the recognition accuracy for eight substrates, each with ten latent fingerprints: white furniture surface, veneered plywood, brushed stainless steel, aluminum foil, "Golden-Oak" veneer, non-metallic matte car body finish, metallic car body finish and blued metal. In comparison to Hildebrandt et al.,1 our evaluation shows a significant reduction of the error rates by 15.8 percent points on brushed stainless steel using the same classifier. This also allows for a successful biometric matching of 3 of the 8 latent fingerprint samples with the corresponding exemplar fingerprint on this particular substrate. For contrast enhancement analysis of classification results we suggest to use known Visual Quality Indexes (VQI)3 as a contrast enhancement quality indicator and discuss our first preliminary results using the exemplary chosen VQI Edge Similarity Score (ESS),4 showing a tendency that higher image differences between a substrate containing a fingerprint and a substrate with a blank surface correlate with a higher recognition accuracy between a latent fingerprint and an exemplar fingerprint. Those first preliminary results support further research into VQIs as contrast enhancement quality indicator for a given feature space.

Comparison of intrinsic dynamics of cytochrome p450 proteins using normal mode analysis

PubMed Central

Dorner, Mariah E; McMunn, Ryan D; Bartholow, Thomas G; Calhoon, Brecken E; Conlon, Michelle R; Dulli, Jessica M; Fehling, Samuel C; Fisher, Cody R; Hodgson, Shane W; Keenan, Shawn W; Kruger, Alyssa N; Mabin, Justin W; Mazula, Daniel L; Monte, Christopher A; Olthafer, Augustus; Sexton, Ashley E; Soderholm, Beatrice R; Strom, Alexander M; Hati, Sanchita

2015-01-01

Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but < 25% sequence identity. Based on their electron transfer protein partner, cytochrome P450 proteins are classified into six broad classes. Traditional methods of pro are based on the canonical paradigm that attributes proteins' function to their three-dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product > 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in Cα atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes. PMID:26130403

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai

2008-07-08

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar tomore » tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.« less

Substrate scope of a dehydrogenase from Sphingomonas species A1 and its potential application in the synthesis of rare sugars and sugar derivatives.

PubMed

Beer, Barbara; Pick, André; Döring, Manuel; Lommes, Petra; Sieber, Volker

2018-07-01

Rare sugars and sugar derivatives that can be obtained from abundant sugars are of great interest to biochemical and pharmaceutical research. Here, we describe the substrate scope of a short-chain dehydrogenase/reductase from Sphingomonas species A1 (SpsADH) in the oxidation of aldonates and polyols. The resulting products are rare uronic acids and rare sugars respectively. We provide insight into the substrate recognition of SpsADH using kinetic analyses, which show that the configuration of the hydroxyl groups adjacent to the oxidized carbon is crucial for substrate recognition. Furthermore, the specificity is demonstrated by the oxidation of d-sorbitol leading to l-gulose as sole product instead of a mixture of d-glucose and l-gulose. Finally, we applied the enzyme to the synthesis of l-gulose from d-sorbitol in an in vitro system using a NADH oxidase for cofactor recycling. This study shows the usefulness of exploring the substrate scope of enzymes to find new enzymatic reaction pathways from renewable resources to value-added compounds. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilderman, P. Ross, E-mail: pwilderman@ucsd.edu; Jang, Hyun-Hee; Malenke, Jael R.

Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in Escherichia coli each of the woodrat proteinsmore » gave the characteristic maximum at 450 nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system. - Highlights: • Three CYP2B enzymes from Neotoma lepida were cloned, engineered, and expressed. • A mix of catalytic and binding assays yields unique results for each enzyme. • Mutational analysis indicates CYP{sub 2}B substrate recognition remains to be clarified. • Reported N. lepida gene sequences allow for larger scale analyses of CYP{sub 2}B enzymes.« less

Structural basis for dynamic mechanism of nitrate/nitrite antiport by NarK

NASA Astrophysics Data System (ADS)

Fukuda, Masahiro; Takeda, Hironori; Kato, Hideaki E.; Doki, Shintaro; Ito, Koichi; Maturana, Andrés D.; Ishitani, Ryuichiro; Nureki, Osamu

2015-05-01

NarK belongs to the nitrate/nitrite porter (NNP) family in the major facilitator superfamily (MFS) and plays a central role in nitrate uptake across the membrane in diverse organisms, including archaea, bacteria, fungi and plants. Although previous studies provided insight into the overall structure and the substrate recognition of NarK, its molecular mechanism, including the driving force for nitrate transport, remained elusive. Here we demonstrate that NarK is a nitrate/nitrite antiporter, using an in vitro reconstituted system. Furthermore, we present the high-resolution crystal structures of NarK from Escherichia coli in the nitrate-bound occluded, nitrate-bound inward-open and apo inward-open states. The integrated structural, functional and computational analyses reveal the nitrate/nitrite antiport mechanism of NarK, in which substrate recognition is coupled to the transport cycle by the concomitant movement of the transmembrane helices and the key tyrosine and arginine residues in the substrate-binding site.

Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10.

PubMed

Swinehart, William E; Henderson, Jeremy C; Jackman, Jane E

2013-08-01

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m1G9 modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m1G9 in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m1G9 methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m1G9 containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility*

PubMed Central

Buechner, Claudia N.; Heil, Korbinian; Michels, Gudrun; Carell, Thomas; Kisker, Caroline; Tessmer, Ingrid

2014-01-01

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5′-3′ helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a “final” verification state, which may then trigger the recruitment of further NER proteins. PMID:24338567

Segregation of anterior temporal regions critical for retrieving names of unique and nonunique entities reflects underlying long-range connectivity

PubMed Central

Mehta, Sonya; Inoue, Kayo; Rudrauf, David; Damasio, Hanna; Tranel, Daniel; Grabowski, Thomas

2015-01-01

Lesion-deficit studies support the hypothesis that the left anterior temporal lobe (ATL) plays a critical role in retrieving names of concrete entities. They further suggest that different regions of the left ATL process different conceptual categories. Here we test the specificity of these relationships and whether the anatomical segregation is related to the underlying organization of white matter connections. We reanalyzed data from a previous lesion study of naming and recognition across five categories of concrete entities. In voxelwise logistic regressions of lesion-deficit associations, we formally incorporated measures of disconnection of long-range association fiber tracts (FTs) and covaried for recognition and non-category specific naming deficits. We also performed fiber tractwise analyses to assess whether damage to specific FTs was preferentially associated with category-selective naming deficits. Damage to the basolateral ATL was associated with naming deficits for both unique (famous faces) and non-unique entities, whereas the damage to the temporal pole was associated with naming deficits for unique entities only. This segregation pattern remained after accounting for comorbid recognition deficits or naming deficits in other categories. The tractwise analyses showed that damage to the uncinate fasciculus was associated with naming impairments for unique entities, while damage to the inferior longitudinal fasciculus was associated with naming impairments for non-unique entities. Covarying for FT transection in voxelwise analyses rendered the cortical association for unique entities more focal. These results are consistent with the partial segregation of brain system support for name retrieval of unique and non-unique entities at both the level of cortical components and underlying white matter fiber bundles. Our study reconciles theoretic accounts of the functional organization of the left ATL by revealing both category-related processing and semantic hub sectors. PMID:26707082

Emotion processing in chimeric faces: hemispheric asymmetries in expression and recognition of emotions.

PubMed

Indersmitten, Tim; Gur, Ruben C

2003-05-01

Since the discovery of facial asymmetries in emotional expressions of humans and other primates, hypotheses have related the greater left-hemiface intensity to right-hemispheric dominance in emotion processing. However, the difficulty of creating true frontal views of facial expressions in two-dimensional photographs has confounded efforts to better understand the phenomenon. We have recently described a method for obtaining three-dimensional photographs of posed and evoked emotional expressions and used these stimuli to investigate both intensity of expression and accuracy of recognizing emotion in chimeric faces constructed from only left- or right-side composites. The participant population included 38 (19 male, 19 female) African-American, Caucasian, and Asian adults. They were presented with chimeric composites generated from faces of eight actors and eight actresses showing four emotions: happiness, sadness, anger, and fear, each in posed and evoked conditions. We replicated the finding that emotions are expressed more intensely in the left hemiface for all emotions and conditions, with the exception of evoked anger, which was expressed more intensely in the right hemiface. In contrast, the results indicated that emotional expressions are recognized more efficiently in the right hemiface, indicating that the right hemiface expresses emotions more accurately. The double dissociation between the laterality of expression intensity and that of recognition efficiency supports the notion that the two kinds of processes may have distinct neural substrates. Evoked anger is uniquely expressed more intensely and accurately on the side of the face that projects to the viewer's right hemisphere, dominant in emotion recognition.

Molecular recognition in protein modification with rhodium metallopeptides

PubMed Central

Ball, Zachary T.

2015-01-01

Chemical manipulation of natural, unengineered proteins is a daunting challenge which tests the limits of reaction design. By combining transition-metal or other catalysts with molecular recognition ideas, it is possible to achieve site-selective protein reactivity without the need for engineered recognition sequences or reactive sites. Some recent examples in this area have used ruthenium photocatalysis, pyridine organocatalysis, and rhodium(II) metallocarbene catalysis, indicating that the fundamental ideas provide opportunities for using diverse reactivity on complex protein substrates and in complex cell-like environments. PMID:25588960

The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux.

PubMed

Sealover, Natalie R; Felts, Bruce; Kuntz, Charles P; Jarrard, Rachel E; Hockerman, Gregory H; Lamb, Patrick W; Barker, Eric L; Henry, L Keith

2016-11-15

The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural characterization of the bacterial proteasome homolog BPH reveals a tetradecameric double-ring complex with unique inner cavity properties.

PubMed

Fuchs, Adrian C D; Maldoner, Lorena; Hipp, Katharina; Hartmann, Marcus D; Martin, Jörg

2018-01-19

Eukaryotic and archaeal proteasomes are paradigms for self-compartmentalizing proteases. To a large extent, their function requires interplay with hexameric ATPases associated with diverse cellular activities (AAA+) that act as substrate unfoldases. Bacteria have various types of self-compartmentalizing proteases; in addition to the proteasome itself, these include the proteasome homolog HslV, which functions together with the AAA+ HslU; the ClpP protease with its partner AAA+ ClpX; and Anbu, a recently characterized ancestral proteasome variant. Previous bioinformatic analysis has revealed a novel bacterial member of the proteasome family Betaproteobacteria proteasome homolog (BPH). Using cluster analysis, we here affirmed that BPH evolutionarily descends from HslV. Crystal structures of the Thiobacillus denitrificans and Cupriavidus metallidurans BPHs disclosed a homo-oligomeric double-ring architecture in which the active sites face the interior of the cylinder. Using small-angle X-ray scattering (SAXS) and electron microscopy averaging, we found that BPH forms tetradecamers in solution, unlike the dodecamers seen in HslV. Although the highly acidic inner surface of BPH was in striking contrast to the cavity characteristics of the proteasome and HslV, a classical proteasomal reaction mechanism could be inferred from the covalent binding of the proteasome-specific inhibitor epoxomicin to BPH. A ligand-bound structure implied that the elongated BPH inner pore loop may be involved in substrate recognition. The apparent lack of a partner unfoldase and other unique features, such as Ser replacing Thr as the catalytic residue in certain BPH subfamilies, suggest a proteolytic function for BPH distinct from those of known bacterial self-compartmentalizing proteases. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural characterization of the bacterial proteasome homolog BPH reveals a tetradecameric double-ring complex with unique inner cavity properties

PubMed Central

Fuchs, Adrian C. D.; Maldoner, Lorena; Hipp, Katharina; Hartmann, Marcus D.; Martin, Jörg

2018-01-01

Eukaryotic and archaeal proteasomes are paradigms for self-compartmentalizing proteases. To a large extent, their function requires interplay with hexameric ATPases associated with diverse cellular activities (AAA+) that act as substrate unfoldases. Bacteria have various types of self-compartmentalizing proteases; in addition to the proteasome itself, these include the proteasome homolog HslV, which functions together with the AAA+ HslU; the ClpP protease with its partner AAA+ ClpX; and Anbu, a recently characterized ancestral proteasome variant. Previous bioinformatic analysis has revealed a novel bacterial member of the proteasome family Betaproteobacteria proteasome homolog (BPH). Using cluster analysis, we here affirmed that BPH evolutionarily descends from HslV. Crystal structures of the Thiobacillus denitrificans and Cupriavidus metallidurans BPHs disclosed a homo-oligomeric double-ring architecture in which the active sites face the interior of the cylinder. Using small-angle X-ray scattering (SAXS) and electron microscopy averaging, we found that BPH forms tetradecamers in solution, unlike the dodecamers seen in HslV. Although the highly acidic inner surface of BPH was in striking contrast to the cavity characteristics of the proteasome and HslV, a classical proteasomal reaction mechanism could be inferred from the covalent binding of the proteasome-specific inhibitor epoxomicin to BPH. A ligand-bound structure implied that the elongated BPH inner pore loop may be involved in substrate recognition. The apparent lack of a partner unfoldase and other unique features, such as Ser replacing Thr as the catalytic residue in certain BPH subfamilies, suggest a proteolytic function for BPH distinct from those of known bacterial self-compartmentalizing proteases. PMID:29183996

Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis.

PubMed

Jiang, Zheng-Yu; Xu, Li-Li; Lu, Meng-Chen; Pan, Yang; Huang, Hao-Ze; Zhang, Xiao-Jin; Sun, Hao-Peng; You, Qi-Dong

2014-12-01

E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates.

PubMed

Yoshida, Hiromi; Yoshihara, Akihide; Ishii, Tomohiko; Izumori, Ken; Kamitori, Shigehiro

2016-12-01

Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.

Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis

NASA Astrophysics Data System (ADS)

Jiang, Zheng-Yu; Xu, Li-Li; Lu, Meng-Chen; Pan, Yang; Huang, Hao-Ze; Zhang, Xiao-Jin; Sun, Hao-Peng; You, Qi-Dong

2014-12-01

E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome

PubMed Central

McGinty, Robert K.; Henrici, Ryan C.; Tan, Song

2014-01-01

The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in higher eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the Ring1B/Bmi1/UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with multiple nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome. PMID:25355358

Role of the Anterior Cingulate Cortex in the Retrieval of Novel Object Recognition Memory after a Long Delay

ERIC Educational Resources Information Center

Pezze, Marie A.; Marshall, Hayley J.; Fone, Kevin C. F.; Cassaday, Helen J.

2017-01-01

Previous in vivo electrophysiological studies suggest that the anterior cingulate cortex (ACgx) is an important substrate of novel object recognition (NOR) memory. However, intervention studies are needed to confirm this conclusion and permanent lesion studies cannot distinguish effects on encoding and retrieval. The interval between encoding and…

New strategies to inhibit KEAP1 and the Cul3-based E3 ubiquitin ligases

PubMed Central

Canning, Peter; Bullock, Alex N.

2014-01-01

E3 ubiquitin ligases that direct substrate proteins to the ubiquitin–proteasome system are promising, though largely unexplored drug targets both because of their function and their remarkable specificity. CRLs [Cullin–RING (really interesting new gene) ligases] are the largest group of E3 ligases and function as modular multisubunit complexes constructed around a Cullin-family scaffold protein. The Cul3-based CRLs uniquely assemble with BTB (broad complex/tramtrack/bric-à-brac) proteins that also homodimerize and perform the role of both the Cullin adapter and the substrate-recognition component of the E3. The most prominent member is the BTB–BACK (BTB and C-terminal Kelch)–Kelch protein KEAP1 (Kelch-like ECH-associated protein 1), a master regulator of the oxidative stress response and a potential drug target for common conditions such as diabetes, Alzheimer's disease and Parkinson's disease. Structural characterization of BTB–Cul3 complexes has revealed a number of critical assembly mechanisms, including the binding of an N-terminal Cullin extension to a bihelical ‘3-box’ at the C-terminus of the BTB domain. Improved understanding of the structure of these complexes should contribute significantly to the effort to develop novel therapeutics targeted to CRL3-regulated pathways. PMID:24450635

Crystal structure of RlmAI: Implications for understanding the 23S rRNA G745/G748-methylation at the macrolide antibiotic-binding site

PubMed Central

Das, Kalyan; Acton, Thomas; Chiang, Yiwen; Shih, Lydia; Arnold, Eddy; Montelione, Gaetano T.

2004-01-01

The RlmA class of enzymes (RlmAI and RlmAII) catalyzes N1-methylation of a guanine base (G745 in Gram-negative and G748 in Gram-positive bacteria) of hairpin 35 of 23S rRNA. We have determined the crystal structure of Escherichia coli RlmAI at 2.8-Å resolution, providing 3D structure information for the RlmA class of RNA methyltransferases. The dimeric protein structure exhibits features that provide new insights into its molecular function. Each RlmAI molecule has a Zn-binding domain, responsible for specific recognition and binding of its rRNA substrate, and a methyltransferase domain. The asymmetric RlmAI dimer observed in the crystal structure has a well defined W-shaped RNA-binding cleft. Two S-adenosyl-l-methionine substrate molecules are located at the two valleys of the W-shaped RNA-binding cleft. The unique shape of the RNA-binding cleft, different from that of known RNA-binding proteins, is highly specific and structurally complements the 3D structure of hairpin 35 of bacterial 23S rRNA. Apart from the hairpin 35, parts of hairpins 33 and 34 also interact with the RlmAI dimer. PMID:14999102

Access channels to the buried active site control substrate specificity in CYP1A P450 enzymes.

PubMed

Urban, Philippe; Truan, Gilles; Pompon, Denis

2015-04-01

A cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity. Time course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps. The global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not. Size discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition. Catalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active site. Copyright © 2014 Elsevier B.V. All rights reserved.

A conserved loop-wedge motif moderates reaction site search and recognition by FEN1.

PubMed

Thompson, Mark J; Gotham, Victoria J B; Ciani, Barbara; Grasby, Jane A

2018-06-07

DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognize opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3'-terminus of a primer strand, which is recognized by breaking the terminal base pair to generate a substrate with a single nucleotide 3'-flap. This recognition event allosterically signals hydrolytic removal of the 5'-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved 'wedge' residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated 'loop-wedge' mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognize irregular DNA structures. These new findings reveal how FEN1 precisely couples 3'-flap verification to function.

16p11.2 Deletion Mice Display Cognitive Deficits in Touchscreen Learning and Novelty Recognition Tasks

ERIC Educational Resources Information Center

Yang, Mu; Lewis, Freeman C.; Sarvi, Michael S.; Foley, Gillian M.; Crawley, Jacqueline N.

2015-01-01

Chromosomal 16p11.2 deletion syndrome frequently presents with intellectual disabilities, speech delays, and autism. Here we investigated the Dolmetsch line of 16p11.2 heterozygous (+/-) mice on a range of cognitive tasks with different neuroanatomical substrates. Robust novel object recognition deficits were replicated in two cohorts of 16p11.2…

Cuticular Hydrocarbons of Tribolium confusum Larvae Mediate Trail Following and Host Recognition in the Ectoparasitoid Holepyris sylvanidis.

PubMed

Fürstenau, Benjamin; Hilker, Monika

2017-09-01

Parasitic wasps which attack insects infesting processed stored food need to locate their hosts hidden inside these products. Their host search is well-known to be guided by host kairomones, perceived via olfaction or contact. Among contact kairomones, host cuticular hydrocarbons (CHCs) may provide reliable information for a parasitoid. However, the chemistry of CHC profiles of hosts living in processed stored food products is largely unknown. Here we showed that the ectoparasitoid Holepyris sylvanidis uses CHCs of its host Tribolium confusum, a worldwide stored product pest, as kairomones for host location and recognition at short range. Chemical analysis of T. confusum larval extracts by gas chromatography coupled with mass spectrometry revealed a rich blend of long-chain (C25-C30) hydrocarbons, including n-alkanes, mono-, and dimethylalkanes. We further studied whether host larvae leave sufficient CHCs on a substrate where they walk along, thus allowing parasitoids to perceive a CHC trail and follow it to their host larvae. We detected 18 CHCs on a substrate that had been exposed to host larvae. These compounds were also found in crude extracts of host larvae and made up about a fifth of the CHC amount extracted. Behavioral assays showed that trails of host CHCs were followed by the parasitoids and reduced their searching time until successful host recognition. Host CHC trails deposited on different substrates were persistent for about a day. Hence, the parasitoid H. sylvanidis exploits CHCs of T. confusum larvae for host finding by following host CHC trails and for host recognition by direct contact with host larvae.

α-Diazo-β-ketonitriles: uniquely reactive substrates for arene and alkene cyclopropanation.

PubMed

Nani, Roger R; Reisman, Sarah E

2013-05-15

An investigation of the intramolecular cyclopropanation reactions of α-diazo-β-ketonitriles is reported. These studies reveal that α-diazo-β-ketonitriles exhibit unique reactivity in their ability to undergo arene cyclopropanation reactions; other similar acceptor-acceptor-substituted diazo substrates instead produce mixtures of C-H insertion and dimerization products. α-Diazo-β-ketonitriles also undergo highly efficient intramolecular cyclopropanation of tri- and tetrasubstituted alkenes. In addition, the α-cyano-α-ketocyclopropane products are demonstrated to serve as substrates for SN2, SN2', and aldehyde cycloaddition reactions.

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. Here we present crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membranemore » proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.« less

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

PubMed Central

McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; Zimmerman, Brandon; Miles, Laura; Beglova, Natalia; Klein, Thomas; Blacklow, Stephen C.

2015-01-01

Summary Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C-terminus. Together, these studies provide new insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential new target for therapeutic modulation of Notch signal transduction in disease. PMID:25747658

A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

NASA Technical Reports Server (NTRS)

Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

1999-01-01

To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

DOE PAGES

McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; ...

2015-03-05

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. Here we present crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membranemore » proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.« less

Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

NASA Astrophysics Data System (ADS)

Deng, Zengqin; Wang, Qing; Liu, Zhao; Zhang, Manfeng; Machado, Ana Carolina Dantas; Chiu, Tsu-Pei; Feng, Chong; Zhang, Qi; Yu, Lin; Qi, Lei; Zheng, Jiangge; Wang, Xu; Huo, Xinmei; Qi, Xiaoxuan; Li, Xiaorong; Wu, Wei; Rohs, Remo; Li, Ying; Chen, Zhongzhou

2015-07-01

Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

Segregation of anterior temporal regions critical for retrieving names of unique and non-unique entities reflects underlying long-range connectivity.

PubMed

Mehta, Sonya; Inoue, Kayo; Rudrauf, David; Damasio, Hanna; Tranel, Daniel; Grabowski, Thomas

2016-02-01

Lesion-deficit studies support the hypothesis that the left anterior temporal lobe (ATL) plays a critical role in retrieving names of concrete entities. They further suggest that different regions of the left ATL process different conceptual categories. Here we test the specificity of these relationships and whether the anatomical segregation is related to the underlying organization of white matter connections. We reanalyzed data from a previous lesion study of naming and recognition across five categories of concrete entities. In voxelwise logistic regressions of lesion-deficit associations, we formally incorporated measures of disconnection of long-range association fiber tracts (FTs) and covaried for recognition and non-category-specific naming deficits. We also performed fiber tractwise analyses to assess whether damage to specific FTs was preferentially associated with category-selective naming deficits. Damage to the basolateral ATL was associated with naming deficits for both unique (famous faces) and non-unique entities, whereas the damage to the temporal pole was associated with naming deficits for unique entities only. This segregation pattern remained after accounting for comorbid recognition deficits or naming deficits in other categories. The tractwise analyses showed that damage to the uncinate fasciculus (UNC) was associated with naming impairments for unique entities, while damage to the inferior longitudinal fasciculus (ILF) was associated with naming impairments for non-unique entities. Covarying for FT transection in voxelwise analyses rendered the cortical association for unique entities more focal. These results are consistent with the partial segregation of brain system support for name retrieval of unique and non-unique entities at both the level of cortical components and underlying white matter fiber bundles. Our study reconciles theoretic accounts of the functional organization of the left ATL by revealing both category-related processing and semantic hub sectors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Recognition Memory zROC Slopes for Items with Correct versus Incorrect Source Decisions Discriminate the Dual Process and Unequal Variance Signal Detection Models

ERIC Educational Resources Information Center

Starns, Jeffrey J.; Rotello, Caren M.; Hautus, Michael J.

2014-01-01

We tested the dual process and unequal variance signal detection models by jointly modeling recognition and source confidence ratings. The 2 approaches make unique predictions for the slope of the recognition memory zROC function for items with correct versus incorrect source decisions. The standard bivariate Gaussian version of the unequal…

Self-face recognition in children with autism spectrum disorders: a near-infrared spectroscopy study.

PubMed

Kita, Yosuke; Gunji, Atsuko; Inoue, Yuki; Goto, Takaaki; Sakihara, Kotoe; Kaga, Makiko; Inagaki, Masumi; Hosokawa, Toru

2011-06-01

It is assumed that children with autism spectrum disorders (ASD) have specificities for self-face recognition, which is known to be a basic cognitive ability for social development. In the present study, we investigated neurological substrates and potentially influential factors for self-face recognition of ASD patients using near-infrared spectroscopy (NIRS). The subjects were 11 healthy adult men, 13 normally developing boys, and 10 boys with ASD. Their hemodynamic activities in the frontal area and their scanning strategies (eye-movement) were examined during self-face recognition. Other factors such as ASD severities and self-consciousness were also evaluated by parents and patients, respectively. Oxygenated hemoglobin levels were higher in the regions corresponding to the right inferior frontal gyrus than in those corresponding to the left inferior frontal gyrus. In two groups of children these activities reflected ASD severities, such that the more serious ASD characteristics corresponded with lower activity levels. Moreover, higher levels of public self-consciousness intensified the activities, which were not influenced by the scanning strategies. These findings suggest that dysfunction in the right inferior frontal gyrus areas responsible for self-face recognition is one of the crucial neural substrates underlying ASD characteristics, which could potentially be used to evaluate psychological aspects such as public self-consciousness. Copyright © 2010 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB_REDO strategies.

PubMed

Rimsa, Vadim; Eadsforth, Thomas C; Joosten, Robbie P; Hunter, William N

2014-02-01

A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB_REDO were coupled with model-map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn2+-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn2+, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1' recognition subsite that suggests specificity towards an acidic substrate.

Substrate degradation by the proteasome: a single-molecule kinetic analysis

PubMed Central

Lu, Ying; Lee, Byung-hoon; King, Randall W; Finley, Daniel; Kirschner, Marc W

2015-01-01

To address how the configuration of conjugated ubiquitins determines the recognition of substrates by the proteasome, we analyzed the degradation kinetics of substrates with chemically defined ubiquitin configurations. Contrary to the view that a tetraubiquitin chain is the minimal signal for efficient degradation, we find that distributing the ubiquitins as diubiquitin chains provides a more efficient signal. To understand how the proteasome actually discriminates among ubiquitin configurations, we developed single-molecule assays that distinguished intermediate steps of degradation kinetically. The level of ubiquitin on a substrate drives proteasome-substrate interaction, whereas the chain structure of ubiquitin affects translocation into the axial channel on the proteasome. Together these two features largely determine the susceptibility of substrates for proteasomal degradation. PMID:25859050

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1-42)

NASA Astrophysics Data System (ADS)

Matsumoto, Erino; Yamauchi, Takahiro; Fukuda, Tomohiro; Miura, Yoshiko

2009-06-01

Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs) on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide-protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

The Potential of Using Brain Images for Authentication

PubMed Central

Zhou, Zongtan; Shen, Hui; Hu, Dewen

2014-01-01

Biometric recognition (also known as biometrics) refers to the automated recognition of individuals based on their biological or behavioral traits. Examples of biometric traits include fingerprint, palmprint, iris, and face. The brain is the most important and complex organ in the human body. Can it be used as a biometric trait? In this study, we analyze the uniqueness of the brain and try to use the brain for identity authentication. The proposed brain-based verification system operates in two stages: gray matter extraction and gray matter matching. A modified brain segmentation algorithm is implemented for extracting gray matter from an input brain image. Then, an alignment-based matching algorithm is developed for brain matching. Experimental results on two data sets show that the proposed brain recognition system meets the high accuracy requirement of identity authentication. Though currently the acquisition of the brain is still time consuming and expensive, brain images are highly unique and have the potential possibility for authentication in view of pattern recognition. PMID:25126604

The potential of using brain images for authentication.

PubMed

Chen, Fanglin; Zhou, Zongtan; Shen, Hui; Hu, Dewen

2014-01-01

Biometric recognition (also known as biometrics) refers to the automated recognition of individuals based on their biological or behavioral traits. Examples of biometric traits include fingerprint, palmprint, iris, and face. The brain is the most important and complex organ in the human body. Can it be used as a biometric trait? In this study, we analyze the uniqueness of the brain and try to use the brain for identity authentication. The proposed brain-based verification system operates in two stages: gray matter extraction and gray matter matching. A modified brain segmentation algorithm is implemented for extracting gray matter from an input brain image. Then, an alignment-based matching algorithm is developed for brain matching. Experimental results on two data sets show that the proposed brain recognition system meets the high accuracy requirement of identity authentication. Though currently the acquisition of the brain is still time consuming and expensive, brain images are highly unique and have the potential possibility for authentication in view of pattern recognition.

Differential recognition of ACE inhibitors in Xenopus laevis oocytes expressing rat PEPT1 and PEPT2.

PubMed

Zhu, T; Chen, X Z; Steel, A; Hediger, M A; Smith, D E

2000-05-01

To examine the mechanism of inhibition of glycylsarcosine (GlySar) transport by quinapril and enalapril, and whether or not angiotensin converting enzyme (ACE) inhibitors are transported by PEPT2 as well as by PEPT1. Xenopus laevis oocytes were cRNA-injected with rat PEPT1 or PEPT2 and the transport kinetics of radiolabeled GlySar were studied in the absence and presence of quinapril and enalapril. The two-microelectrode voltage-clamp technique was also performed to probe the electrogenic uptake of captopril, quinapril and enalapril. Kinetic analyses demonstrated that quinapril inhibited the uptake of GlySar in a noncompetitive manner in Xenopus oocytes injected with PEPT1 or PEPT2 (Ki = 0.8 or 0.4 mM, respectively). In contrast, a competitive interaction was observed between GlySar and enalapril (Ki = 10.8 mM for PEPT1 or 4.3 mM for PEPT2). Most significantly, captopril and enalapril, but not quinapril, induced inwardly-directed currents in both PEPT1- and PEPT2-expressed oocytes. These results are unique in providing direct evidence for the substrate recognition and transport of some ACE inhibitors by the high- and low-affinity oligopeptide transporters. Our findings point to differences between PEPT1 and PEPT2 in their affinity to, rather than in their specificity for, ACE inhibitors.

Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1).

PubMed

Asciutto, Eliana K; Pochapsky, Thomas C

2018-04-27

Cytochrome P450 cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1 H- 15 N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Allosteric response and substrate sensitivity in peptide binding of the signal recognition particle.

PubMed

Wang, Connie Y; Miller, Thomas F

2014-10-31

We characterize the conformational dynamics and substrate selectivity of the signal recognition particle (SRP) using a thermodynamic free energy cycle approach and microsecond timescale molecular dynamics simulations. The SRP is a central component of the co-translational protein targeting machinery that binds to the N-terminal signal peptide (SP) of nascent proteins. We determined the shift in relative conformational stability of the SRP upon substrate binding to quantify allosteric coupling between SRP domains. In particular, for dipeptidyl aminopeptidase, an SP that is recognized by the SRP for co-translational targeting, it is found that substrate binding induces substantial changes in the SRP toward configurations associated with targeting of the nascent protein, and it is found that the changes are modestly enhanced by a mutation that increases the hydrophobicity of the SP. However, for alkaline phosphatase, an SP that is recognized for post-translational targeting, substrate binding induces the reverse change in the SRP conformational distribution away from targeting configurations. Microsecond timescale trajectories reveal the intrinsic flexibility of the SRP conformational landscape and provide insight into recent single molecule studies by illustrating that 10-nm lengthscale changes between FRET pairs occur via the rigid-body movement of SRP domains connected by the flexible linker region. In combination, these results provide direct evidence for the hypothesis that substrate-controlled conformational switching in the SRP provides a mechanism for discriminating between different SPs and for connecting substrate binding to downstream steps in the protein targeting pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A unique memory process modulated by emotion underpins successful odor recognition and episodic retrieval in humans

PubMed Central

Saive, Anne-Lise; Royet, Jean-Pierre; Ravel, Nadine; Thévenet, Marc; Garcia, Samuel; Plailly, Jane

2014-01-01

We behaviorally explore the link between olfaction, emotion and memory by testing the hypothesis that the emotion carried by odors facilitates the memory of specific unique events. To investigate this idea, we used a novel behavioral approach inspired by a paradigm developed by our team to study episodic memory in a controlled and as ecological as possible way in humans. The participants freely explored three unique and rich laboratory episodes; each episode consisted of three unfamiliar odors (What) positioned at three specific locations (Where) within a visual context (Which context). During the retrieval test, which occurred 24–72 h after the encoding, odors were used to trigger the retrieval of the complex episodes. The participants were proficient in recognizing the target odors among distractors and retrieving the visuospatial context in which they were encountered. The episodic nature of the task generated high and stable memory performances, which were accompanied by faster responses and slower and deeper breathing. Successful odor recognition and episodic memory were not related to differences in odor investigation at encoding. However, memory performances were influenced by the emotional content of the odors, regardless of odor valence, with both pleasant and unpleasant odors generating higher recognition and episodic retrieval than neutral odors. Finally, the present study also suggested that when the binding between the odors and the spatio-contextual features of the episode was successful, the odor recognition and the episodic retrieval collapsed into a unique memory process that began as soon as the participants smelled the odors. PMID:24936176

Diversity in peptide recognition by the SH2 domain of SH2B1.

PubMed

McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

2018-02-01

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein-protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate-1 and -2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C-terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high-resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand-binding interface of SH2B1 with high specificity. © 2017 Wiley Periodicals, Inc.

A highly sensitive biological detection substrate based on TiO2 nanowires supporting gold nanoparticles

NASA Astrophysics Data System (ADS)

Zeng, Yuan; Tan, Hai-jun; Cheng, Xiu-Lan; Chen, Rui; Wang, Ying

2011-12-01

Surface enhanced Raman scattering (SERS) has attracted widespread concern in the field of bioassay because it can enhance normally weak Raman signal by several orders of magnitude and facilitate the highly sensitive detection of molecules. Conventional SERS substrates are prepared by placing metal nanoparticles on a planar surface. Here we show a unique SERS substrate stacked by disordered TiO2 nanowires (TiO2-NWs) supportig gold nanocrystals. The structure can be easily fabricated by chemical synthesis at low cost. The COMSOL model simulation shows the designed SERS substrate is capable of output high Local Field Enhancement (LFE) in the Near Infrared region (NIR) that is the optimal wavelength in bio-detection because of both the unique coupling enhancement effect amony nearby Au nanocrystals on TiO2-NWs and the Suface Plasmon Resonance (SPR) effect of TiO2 -NWs. The as-prepared transparent and freestanding SERS substrate is capable of detecting extremely low concentration R6G molecular, showing much higher Raman signal because of the extremely large surface area and the uniqueTiO2-NWs self-assemblied by Au nanocrystals. These results provide a new approach to ultrasensitive bioassay device.

The recognition and interpretation of micro-particle impacts on space craft surfaces

NASA Astrophysics Data System (ADS)

Kearsley, Anton

Modern analysis instruments now allow the rapid examination of returned spacecraft surfaces, enabling the location and identification of impact features, and the attribution of their impactor origins. This paper describes application of novel electron, ion and micro-X-ray Fluorescence techniques to impacts on diverse compositions of substrate, including solar cell glass, poly-sulfone and fluoro-polymer-impregnated glass fibre composites, multilayer insulation foils, aluminium and titanium alloys. Examples will include two generations of solar cells and stiffener materials from the Hubble Space Telescope (HST), Beta-cloth from the NASA Mir-Trek cover blanket, aluminised Kapton foils from the Japanese Space Flyer Unit (SFU) and the European Retrievable Carrier (EuReCa), Al-alloy plates from the Long Duration Exposure Facility (LDEF), Al foils from the NASA Stardust mission, Al-alloy and Zinc orthotitanate (ZOT) painted alloy plate from HST, and titanium alloys from a re-entered titanium pressure tank. Each type of spacecraft surface poses unique problems of analysis, especially in the recognition of extraneous signatures from the impacting particle, especially if a complex chemical composition is already present in the target. For example, solar cells provide an excellent capture and analysis medium for monitoring fluxes of micrometre-scale orbital debris from solid rocket motor firings in low Earth orbit. However, they provide a hard and dense capture medium upon which substantial modification of the impactor may occur, making the precise identification of micrometeoroid components difficult. Unfortunately, extensive spallation by larger (> 100 micrometre) particle impacts on the thin and brittle structure of solar cells also usually results in complete loss of impactor signature. Although thick alloy surfaces may prevent complete impact penetration, the analysis of particle residues within their deep concavity has proven difficult, until the recent introduction of new high-efficiency energy dispersive X-ray (EDX) detectors. Along with Particle Induced X-ray Emission and micro-XRF imaging systems, the new EDX detectors allow recognition of subtle trace quantities of residue from even millimetre-scale craters and thin foil penetrations. Data from these differing substrates and techniques need to be integrated if we are to fully document micro-particle populations from the wide range of natural (cometary and asteroidal, i.e. micrometeoroid) and artificial (i.e. orbital debris) sources.

The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition

PubMed Central

Wienk, Hans; Slootweg, Jack C.; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E.

2013-01-01

To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition. PMID:23661679

DNA-Templated Pd Conductive Metallic Nanowires

NASA Astrophysics Data System (ADS)

Nguyen, K.; Monteverde, M.; Lyonnais, S.; Campidelli, S.; Bourgoin, J.-Ph.; Filoramo, A.

2008-10-01

Because of its unique recognition properties, its size and the sub-nanometric resolution, DNA is of particular interest for positioning and organizing nanomaterials. However, in DNA-directed nanoelectronic it can be envisioned to use DNA not only as a positioning scaffold, but also as a support for the conducting element. To ensure this function a metallization process is necessary and among the various DNA metallization methods the Pd based ones are of particular interest for carbon nanotube transistor connections. In this field, the major drawback of the existing methods is the fast kinetics of the process which lead to a stochastic growth. Here, we present a novel approach to DNA Pd metalization where the DNA molecule is previously deposited on a dry substrate in a typical nanodevice configuration. In our approach the progressive growth of nanowires is achieved by the slow and selective precipitation of PdO, followed by a subsequent reduction step. Thanks to this strategy we fabricated homogeneous, continuous and conductive Pd nanowires on the DNA scaffolds of very thin diameter (20-25 nm).

Crystal Structures of the SpoIID Lytic Transglycosylases Essential for Bacterial Sporulation.

PubMed

Nocadello, Salvatore; Minasov, George; Shuvalova, Ludmilla S; Dubrovska, Ievgeniia; Sabini, Elisabetta; Anderson, Wayne F

2016-07-15

Bacterial spores are the most resistant form of life known on Earth and represent a serious problem for (i) bioterrorism attack, (ii) horizontal transmission of microbial pathogens in the community, and (iii) persistence in patients and in a nosocomial environment. Stage II sporulation protein D (SpoIID) is a lytic transglycosylase (LT) essential for sporulation. The LT superfamily is a potential drug target because it is active in essential bacterial processes involving the peptidoglycan, which is unique to bacteria. However, the absence of structural information for the sporulation-specific LT enzymes has hindered mechanistic understanding of SpoIID. Here, we report the first crystal structures with and without ligands of the SpoIID family from two community relevant spore-forming pathogens, Bacillus anthracis and Clostridium difficile. The structures allow us to visualize the overall architecture, characterize the substrate recognition model, identify critical residues, and provide the structural basis for catalysis by this new family of enzymes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hornemann, Andrea, E-mail: andrea.hornemann@ptb.de; Hoehl, Arne, E-mail: arne.hoehl@ptb.de; Ulm, Gerhard, E-mail: gerhard.ulm@ptb.de

Bio-diagnostic assays of high complexity rely on nanoscaled assay recognition elements that can provide unique selectivity and design-enhanced sensitivity features. High throughput performance requires the simultaneous detection of various analytes combined with appropriate bioassay components. Nanoparticle induced sensitivity enhancement, and subsequent multiplexed capability Surface-Enhanced InfraRed Absorption (SEIRA) assay formats are fitting well these purposes. SEIRA constitutes an ideal platform to isolate the vibrational signatures of targeted bioassay and active molecules. The potential of several targeted biolabels, here fluorophore-labeled antibody conjugates, chemisorbed onto low-cost biocompatible gold nano-aggregates substrates have been explored for their use in assay platforms. Dried films were analyzedmore » by synchrotron radiation based FTIR/SEIRA spectro-microscopy and the resulting complex hyperspectral datasets were submitted to automated statistical analysis, namely Principal Components Analysis (PCA). The relationships between molecular fingerprints were put in evidence to highlight their spectral discrimination capabilities. We demonstrate that robust spectral encoding via SEIRA fingerprints opens up new opportunities for fast, reliable and multiplexed high-end screening not only in biodiagnostics but also in vitro biochemical imaging.« less

Robust Optical Recognition of Cursive Pashto Script Using Scale, Rotation and Location Invariant Approach

PubMed Central

Ahmad, Riaz; Naz, Saeeda; Afzal, Muhammad Zeshan; Amin, Sayed Hassan; Breuel, Thomas

2015-01-01

The presence of a large number of unique shapes called ligatures in cursive languages, along with variations due to scaling, orientation and location provides one of the most challenging pattern recognition problems. Recognition of the large number of ligatures is often a complicated task in oriental languages such as Pashto, Urdu, Persian and Arabic. Research on cursive script recognition often ignores the fact that scaling, orientation, location and font variations are common in printed cursive text. Therefore, these variations are not included in image databases and in experimental evaluations. This research uncovers challenges faced by Arabic cursive script recognition in a holistic framework by considering Pashto as a test case, because Pashto language has larger alphabet set than Arabic, Persian and Urdu. A database containing 8000 images of 1000 unique ligatures having scaling, orientation and location variations is introduced. In this article, a feature space based on scale invariant feature transform (SIFT) along with a segmentation framework has been proposed for overcoming the above mentioned challenges. The experimental results show a significantly improved performance of proposed scheme over traditional feature extraction techniques such as principal component analysis (PCA). PMID:26368566

Chemically mediated species recognition in closely related Podarcis wall lizards.

PubMed

Barbosa, Diana; Font, Enrique; Desfilis, Ester; Carretero, Miguel A

2006-07-01

In many animals, chemical signals play an important role in species recognition and may contribute to reproductive isolation and speciation. The Iberian lizards of the genus Podarcis, with up to nine currently recognized lineages that are often sympatric, are highly chemosensory and provide an excellent model for the study of chemically mediated species recognition in closely related taxa. In this study, we tested the ability of male and female lizards of two sister species with widely overlapping distribution ranges (Podarcis bocagei and P. hispanica type 1) to discriminate between conspecific and heterospecific mates by using only substrate-borne chemical cues. We scored the number of tongue flicks directed at the paper substrate by each individual in a terrarium previously occupied by a conspecific or a heterospecific lizard of the opposite sex. Results show that males of P. bocagei and P. hispanica type 1 are capable of discriminating chemically between conspecifics and heterospecifics of the opposite sex, but females are not. These results suggest that differences in female, but not male, chemical cues may underlie species recognition and contribute to reproductive isolation in these species. The apparent inability of females to discriminate conspecific from heterospecific males, which is not because of reduced baseline exploration rates, is discussed in the context of sexual selection theory and species discrimination.

The Response Dynamics of Recognition Memory: Sensitivity and Bias

ERIC Educational Resources Information Center

Koop, Gregory J.; Criss, Amy H.

2016-01-01

Advances in theories of memory are hampered by insufficient metrics for measuring memory. The goal of this paper is to further the development of model-independent, sensitive empirical measures of the recognition decision process. We evaluate whether metrics from continuous mouse tracking, or response dynamics, uniquely identify response bias and…

Highly sensitive surface-enhanced Raman scattering substrate made from superaligned carbon nanotubes.

PubMed

Sun, Yinghui; Liu, Kai; Miao, Jiao; Wang, Zheyao; Tian, Baozhong; Zhang, Lina; Li, Qunqing; Fan, Shoushan; Jiang, Kaili

2010-05-12

Surface-enhanced Raman scattering (SERS) has attracted wide attention because it can enhance normally weak Raman signal by several orders of magnitude and facilitate the sensitive detection of molecules. Conventional SERS substrates are constructed by placing metal nanoparticles on a planar surface. Here we show that, if the planar surface was substituted by a unique nanoporous surface, the enhancement effect can be dramatically improved. The nanoporous surface can be easily fabricated in batches and at low costs by cross stacking superaligned carbon nanotube films. The as-prepared transparent and freestanding SERS substrate is capable of detecting ambient trinitrotoluene vapor, showing much higher Raman enhancement than ordinary planar substrates because of the extremely large surface area and the unique zero-dimensional at one-dimensional nanostructure. These results not only provide a new approach to ultrasensitive SERS substrates, but also are helpful for improving the fundamental understanding of SERS phenomena.

Facial Emotion Recognition Impairments are Associated with Brain Volume Abnormalities in Individuals with HIV

PubMed Central

Clark, Uraina S.; Walker, Keenan A.; Cohen, Ronald A.; Devlin, Kathryn N.; Folkers, Anna M.; Pina, Mathew M.; Tashima, Karen T.

2015-01-01

Impaired facial emotion recognition abilities in HIV+ patients are well documented, but little is known about the neural etiology of these difficulties. We examined the relation of facial emotion recognition abilities to regional brain volumes in 44 HIV-positive (HIV+) and 44 HIV-negative control (HC) adults. Volumes of structures implicated in HIV− associated neuropathology and emotion recognition were measured on MRI using an automated segmentation tool. Relative to HC, HIV+ patients demonstrated emotion recognition impairments for fearful expressions, reduced anterior cingulate cortex (ACC) volumes, and increased amygdala volumes. In the HIV+ group, fear recognition impairments correlated significantly with ACC, but not amygdala volumes. ACC reductions were also associated with lower nadir CD4 levels (i.e., greater HIV-disease severity). These findings extend our understanding of the neurobiological substrates underlying an essential social function, facial emotion recognition, in HIV+ individuals and implicate HIV-related ACC atrophy in the impairment of these abilities. PMID:25744868

Specificity of Processing α-glucosidase I is guided by the substrate conformation: crystallographic and in silico studies.

PubMed

Barker, Megan K; Rose, David R

2013-05-10

The enzyme “GluI” is key to the synthesis of critical glycoproteins in the cell. We have determined the structure of GluI, and modeled binding with its unique sugar substrate. The specificity of this interaction derives from a unique conformation of the substrate. Understanding the mechanism of the enzyme is of basic importance and relevant to potential development of antiviral inhibitors. Processing α-glucosidase I (GluI) is a key member of the eukaryotic N-glycosylation processing pathway, selectively catalyzing the first glycoprotein trimming step in the endoplasmic reticulum. Inhibition of GluI activity impacts the infectivity of enveloped viruses; however, despite interest in this protein from a structural, enzymatic, and therapeutic standpoint, little is known about its structure and enzymatic mechanism in catalysis of the unique glycan substrate Glc3Man9GlcNAc2. The first structural model of eukaryotic GluI is here presented at 2-Å resolution. Two catalytic residues are proposed, mutations of which result in catalytically inactive, properly folded protein. Using Autodocking methods with the known substrate and inhibitors as ligands, including a novel inhibitor characterized in this work, the active site of GluI was mapped. From these results, a model of substrate binding has been formulated, which is most likely conserved in mammalian GluI.

The Structure and Specificity of the Type III Secretion System Effector NleC Suggest a DNA Mimicry Mechanism of Substrate Recognition

PubMed Central

2015-01-01

Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation. PMID:25040221

High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB-REDO strategies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rimsa, Vadim; Eadsforth, Thomas C.; Joosten, Robbie P.

2014-02-01

The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase. A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB-REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previouslymore » only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn{sup 2+}-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn{sup 2+}, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.« less

Real time biometric surveillance with gait recognition

NASA Astrophysics Data System (ADS)

Mohapatra, Subasish; Swain, Anisha; Das, Manaswini; Mohanty, Subhadarshini

2018-04-01

Bio metric surveillance has become indispensable for every system in the recent years. The contribution of bio metric authentication, identification, and screening purposes are widely used in various domains for preventing unauthorized access. A large amount of data needs to be updated, segregated and safeguarded from malicious software and misuse. Bio metrics is the intrinsic characteristics of each individual. Recently fingerprints, iris, passwords, unique keys, and cards are commonly used for authentication purposes. These methods have various issues related to security and confidentiality. These systems are not yet automated to provide the safety and security. The gait recognition system is the alternative for overcoming the drawbacks of the recent bio metric based authentication systems. Gait recognition is newer as it hasn't been implemented in the real-world scenario so far. This is an un-intrusive system that requires no knowledge or co-operation of the subject. Gait is a unique behavioral characteristic of every human being which is hard to imitate. The walking style of an individual teamed with the orientation of joints in the skeletal structure and inclinations between them imparts the unique characteristic. A person can alter one's own external appearance but not skeletal structure. These are real-time, automatic systems that can even process low-resolution images and video frames. In this paper, we have proposed a gait recognition system and compared the performance with conventional bio metric identification systems.

Three-dimensional Structure of Saccharomyces Invertase

PubMed Central

Sainz-Polo, M. Angela; Ramírez-Escudero, Mercedes; Lafraya, Alvaro; González, Beatriz; Marín-Navarro, Julia; Polaina, Julio; Sanz-Aparicio, Julia

2013-01-01

Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv) and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Å resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition. PMID:23430743

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

DOE PAGES

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng; ...

2016-03-31

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Infrared/Terahertz Double Resonance for Chemical Remote Sensing: Signatures and Performance Predictions

DTIC Science & Technology

2011-01-01

remote sensing , such as Fourier-transform infrared spectroscopy, has limited recognition specificity because of atmospheric pressure broadening. Active interrogation techniques promise much greater chemical recognition that can overcome the limits imposed by atmospheric pressure broadening. Here we introduce infrared - terahertz (IR/THz) double resonance spectroscopy as an active means of chemical remote sensing that retains recognition specificity through rare, molecule-unique coincidences between IR molecular absorption and a line-tunable CO2

Design method of ARM based embedded iris recognition system

NASA Astrophysics Data System (ADS)

Wang, Yuanbo; He, Yuqing; Hou, Yushi; Liu, Ting

2008-03-01

With the advantages of non-invasiveness, uniqueness, stability and low false recognition rate, iris recognition has been successfully applied in many fields. Up to now, most of the iris recognition systems are based on PC. However, a PC is not portable and it needs more power. In this paper, we proposed an embedded iris recognition system based on ARM. Considering the requirements of iris image acquisition and recognition algorithm, we analyzed the design method of the iris image acquisition module, designed the ARM processing module and its peripherals, studied the Linux platform and the recognition algorithm based on this platform, finally actualized the design method of ARM-based iris imaging and recognition system. Experimental results show that the ARM platform we used is fast enough to run the iris recognition algorithm, and the data stream can flow smoothly between the camera and the ARM chip based on the embedded Linux system. It's an effective method of using ARM to actualize portable embedded iris recognition system.

Biofragments: An Approach towards Predicting Protein Function Using Biologically Related Fragments and its Application to Mycobacterium tuberculosis CYP126

PubMed Central

Hudson, Sean A; Mashalidis, Ellene H; Bender, Andreas; McLean, Kirsty J; Munro, Andrew W; Abell, Chris

2014-01-01

We present a novel fragment-based approach that tackles some of the challenges for chemical biology of predicting protein function. The general approach, which we have termed biofragments, comprises two key stages. First, a biologically relevant fragment library (biofragment library) can be designed and constructed from known sets of substrate-like ligands for a protein class of interest. Second, the library can be screened for binding to a novel putative ligand-binding protein from the same or similar class, and the characterization of hits provides insight into the basis of ligand recognition, selectivity, and function at the substrate level. As a proof-of-concept, we applied the biofragments approach to the functionally uncharacterized Mycobacterium tuberculosis (Mtb) cytochrome P450 isoform, CYP126. This led to the development of a tailored CYP biofragment library with notable 3D characteristics and a significantly higher screening hit rate (14 %) than standard drug-like fragment libraries screened previously against Mtb CYP121 and 125 (4 % and 1 %, respectively). Biofragment hits were identified that make both substrate-like type-I and inhibitor-like type-II interactions with CYP126. A chemical-fingerprint-based substrate model was built from the hits and used to search a virtual TB metabolome, which led to the discovery that CYP126 has a strong preference for the recognition of aromatics and substrate-like type-I binding of chlorophenol moieties within the active site near the heme. Future catalytic analyses will be focused on assessing CYP126 for potential substrate oxidative dehalogenation. PMID:24677424

Ubiquitin-dependent Protein Degradation at the Yeast Endoplasmic Reticulum and Nuclear Envelope

PubMed Central

Zattas, Dimitrios; Hochstrasser, Mark

2014-01-01

The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and secreted proteins are inserted into or across cell membranes. Its membrane bilayer and luminal compartments provide a favorable environment for the folding and assembly of thousands of newly synthesized proteins. However, protein folding is intrinsically error-prone, and various stress conditions can further increase levels of protein misfolding and damage, particularly in the ER, which can lead to cellular dysfunction and disease. The ubiquitin-proteasome system (UPS) is responsible for the selective destruction of a vast array of protein substrates, either for protein quality control or to allow rapid changes in the levels of specific regulatory proteins. In this review, we will focus on the components and mechanisms of ER-associated protein degradation (ERAD), an important branch of the UPS. ER membranes extend from subcortical regions of the cell to the nuclear envelope, with its continuous outer and inner membranes; the nuclear envelope is a specialized subdomain of the ER. ERAD presents additional challenges to the UPS beyond those faced with soluble substrates of the cytoplasm and nucleus. These include recognition of sugar modifications that occur in the ER, retrotranslocation of proteins across the membrane bilayer, and transfer of substrates from the ER extraction machinery to the proteasome. Here we review characteristics of ERAD substrate degradation signals (degrons), mechanisms underlying substrate recognition and processing by the ERAD machinery, and ideas on the still unresolved problem of how substrate proteins are moved across and extracted from the ER membrane. PMID:25231236

Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism

PubMed Central

Nasuno, Ryo; Hirano, Yoshinori; Itoh, Takafumi; Hakoshima, Toshio; Hibi, Takao; Takagi, Hiroshi

2013-01-01

Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog l-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the l-proline and l-arginine metabolism by acetylating l-Δ1-pyrroline-5-carboxylate, leading to the l-arginine–dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-l-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA–binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the l-Δ1-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs. PMID:23818613

Enhancing the efficiency of sortase-mediated ligations through nickel-peptide complex formation.

PubMed

David Row, R; Roark, Travis J; Philip, Marina C; Perkins, Lorena L; Antos, John M

2015-08-14

A modified sortase A recognition motif containing a masked Ni(2+)-binding peptide was employed to boost the efficiency of sortase-catalyzed ligation reactions. Deactivation of the Ni(2+)-binding peptide using a Ni(2+) additive improved reaction performance at low to equimolar ratios of the glycine amine nucleophile and sortase substrate. The success of this approach was demonstrated with both peptide and protein substrates.

Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hui; Klein, Michael G.; Snell, Gyorgy

Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structuremore » reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.« less

Delayed Self-Recognition in Autism: A Unique Difficulty?

ERIC Educational Resources Information Center

Dunphy-Lelii, Sarah; Wellman, Henry M.

2012-01-01

Achieving a sense of self is a crucial task of ordinary development. With which aspects of self do children with autism have particular difficulty? Two prior studies concluded that children with autism are unimpaired in delayed self-recognition; we confirm and clarify this conclusion by examining it in conjunction with another key aspect of self…

Mechanisms and Neural Basis of Object and Pattern Recognition: A Study with Chess Experts

ERIC Educational Resources Information Center

Bilalic, Merim; Langner, Robert; Erb, Michael; Grodd, Wolfgang

2010-01-01

Comparing experts with novices offers unique insights into the functioning of cognition, based on the maximization of individual differences. Here we used this expertise approach to disentangle the mechanisms and neural basis behind two processes that contribute to everyday expertise: object and pattern recognition. We compared chess experts and…

Predicting the Accuracy of Facial Affect Recognition: The Interaction of Child Maltreatment and Intellectual Functioning

ERIC Educational Resources Information Center

Shenk, Chad E.; Putnam, Frank W.; Noll, Jennie G.

2013-01-01

Previous research demonstrates that both child maltreatment and intellectual performance contribute uniquely to the accurate identification of facial affect by children and adolescents. The purpose of this study was to extend this research by examining whether child maltreatment affects the accuracy of facial recognition differently at varying…

Evidence for Early Morphological Decomposition in Visual Word Recognition

ERIC Educational Resources Information Center

Solomyak, Olla; Marantz, Alec

2010-01-01

We employ a single-trial correlational MEG analysis technique to investigate early processing in the visual recognition of morphologically complex words. Three classes of affixed words were presented in a lexical decision task: free stems (e.g., taxable), bound roots (e.g., tolerable), and unique root words (e.g., vulnerable, the root of which…

An ancient protein-DNA interaction underlying metazoan sex determination.

PubMed

Murphy, Mark W; Lee, John K; Rojo, Sandra; Gearhart, Micah D; Kurahashi, Kayo; Banerjee, Surajit; Loeuille, Guy-André; Bashamboo, Anu; McElreavey, Kenneth; Zarkower, David; Aihara, Hideki; Bardwell, Vivian J

2015-06-01

DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. Here we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are used in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.

An ancient protein-DNA interaction underlying metazoan sex determination

DOE PAGES

Murphy, Mark W.; Lee, John K.; Rojo, Sandra; ...

2015-05-25

DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. In this paper, we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are usedmore » in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Finally, our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.« less

An ancient protein-DNA interaction underlying metazoan sex determination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Mark W.; Lee, John K.; Rojo, Sandra

DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. In this paper, we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are usedmore » in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Finally, our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.« less

Structural and biochemical studies of a fluoroacetyl-CoA-specific thioesterase reveal a molecular basis for fluorine selectivity.

PubMed

Weeks, Amy M; Coyle, Scott M; Jinek, Martin; Doudna, Jennifer A; Chang, Michelle C Y

2010-11-02

We have initiated a broad-based program aimed at understanding the molecular basis of fluorine specificity in enzymatic systems, and in this context, we report crystallographic and biochemical studies on a fluoroacetyl-coenzyme A (CoA) specific thioesterase (FlK) from Streptomyces cattleya. Our data establish that FlK is competent to protect its host from fluoroacetate toxicity in vivo and demonstrate a 10(6)-fold discrimination between fluoroacetyl-CoA (k(cat)/K(M) = 5 × 10⁷ M⁻¹ s⁻¹) and acetyl-CoA (k(cat)/K(M) = 30 M⁻¹ s⁻¹) based on a single fluorine substitution that originates from differences in both substrate reactivity and binding. We show that Thr 42, Glu 50, and His 76 are key catalytic residues and identify several factors that influence substrate selectivity. We propose that FlK minimizes interaction with the thioester carbonyl, leading to selection against acetyl-CoA binding that can be recovered in part by new C═O interactions in the T42S and T42C mutants. We hypothesize that the loss of these interactions is compensated by the entropic driving force for fluorinated substrate binding in a hydrophobic binding pocket created by a lid structure, containing Val 23, Leu 26, Phe 33, and Phe 36, that is not found in other structurally characterized members of this superfamily. We further suggest that water plays a critical role in fluorine specificity based on biochemical and structural studies focused on the unique Phe 36 "gate" residue, which functions to exclude water from the active site. Taken together, the findings from these studies offer molecular insights into organofluorine recognition and design of fluorine-specific enzymes.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

PubMed

Katsu, Kenjiro; Suzuki, Rintaro; Tsuchiya, Wataru; Inagaki, Noritoshi; Yamazaki, Toshimasa; Hisano, Tomomi; Yasui, Yasuo; Komori, Toshiyuki; Koshio, Motoyuki; Kubota, Seiji; Walker, Amanda R; Furukawa, Kiyoshi; Matsui, Katsuhiro

2017-12-11

Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F 2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Neuroanatomical substrates involved in unrelated false facial recognition.

PubMed

Ronzon-Gonzalez, Eliane; Hernandez-Castillo, Carlos R; Pasaye, Erick H; Vaca-Palomares, Israel; Fernandez-Ruiz, Juan

2017-11-22

Identifying faces is a process central for social interaction and a relevant factor in eyewitness theory. False recognition is a critical mistake during an eyewitness's identification scenario because it can lead to a wrongful conviction. Previous studies have described neural areas related to false facial recognition using the standard Deese/Roediger-McDermott (DRM) paradigm, triggering related false recognition. Nonetheless, misidentification of faces without trying to elicit false memories (unrelated false recognition) in a police lineup could involve different cognitive processes, and distinct neural areas. To delve into the neural circuitry of unrelated false recognition, we evaluated the memory and response confidence of participants while watching faces photographs in an fMRI task. Functional activations of unrelated false recognition were identified by contrasting the activation on this condition vs. the activations related to recognition (hits) and correct rejections. The results identified the right precentral and cingulate gyri as areas with distinctive activations during false recognition events suggesting a conflict resulting in a dysfunction during memory retrieval. High confidence suggested that about 50% of misidentifications may be related to an unconscious process. These findings add to our understanding of the construction of facial memories and its biological basis, and the fallibility of the eyewitness testimony.

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition.

PubMed

Woodcock, Clayton B; Yakubov, Aziz B; Reich, Norbert O

2017-08-01

Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K m , k cat , k methylation , and K d for single-stranded and hemimethylated substrates, revealing discrimination of 10 7 -fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

Shin-Etsu super-high-flat substrate for FPD panel photomask

NASA Astrophysics Data System (ADS)

Ishitsuka, Youkou; Harada, Daijitsu; Watabe, Atsushi; Takeuchi, Masaki

2017-07-01

Recently, high-resolution exposure machine has been developed for production of high-definition (HD) panels, and higher-flat photomask substrates for FPD is being expected for panel makers to produce HD panels. In this presentation, we introduce about Shin-Etsu's advanced technique of producing super-high-flat photomask substrates. Shin-Etsu has developed surface polishing and planarization technology with No.1-quality-IC photomask substrates. Our most advanced IC photomask substrates have gained the highest estimation and appreciation from our customers because of their surface quality (non-defect surface without sub-0.1um size defects) and ultimate flatness (sub-0.1um order having achieved). By scaling up those IC photomask substrate technologies and developing unique large-size processing technologies, we have achieved creating high-flat large substrates, even G10-photomask size as well as regular G6-G8 photomask size. The core technology is that the surface shape of the substrate is completely controlled by the unique method. For example, we can regularly produce a substrate with its flatness of triple 5ums; front side flatness, back side flatness and total thickness variation are all less than 5μm. Furthermore, we are able to supply a substrate with its flatness of triple 3ums for G6-photomask size advanced grade, believed to be needed in near future.

Interfacial metal and antibody recognition.

PubMed

Zhou, Tongqing; Hamer, Dean H; Hendrickson, Wayne A; Sattentau, Quentin J; Kwong, Peter D

2005-10-11

The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subsequent steps required for viral entry. Here, we use surface-plasmon resonance to show that Q425 requires calcium for recognition of CD4. Specifically, Q425 binding of calcium resulted in a 55,000-fold enhancement in affinity for CD4. X-ray crystallographic analyses of Q425 in the presence of Ca(2+), Ba(2+), or EDTA revealed an exposed metal-binding site, partially coordinated by five atoms contributed from four antibody complementarity-determining regions. The results suggest that Q425 recognition of CD4 involves direct ligation of antigen by the Q425-held calcium, with calcium binding each ligating atom of CD4 with approximately 1.5 kcal/mol of binding energy. This energetic contribution, which is greater than that from a typical protein atom, demonstrates how interfacial metal ligation can play a unique role in antigen recognition.

Interfacial metal and antibody recognition

PubMed Central

Zhou, Tongqing; Hamer, Dean H.; Hendrickson, Wayne A.; Sattentau, Quentin J.; Kwong, Peter D.

2005-01-01

The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subsequent steps required for viral entry. Here, we use surface-plasmon resonance to show that Q425 requires calcium for recognition of CD4. Specifically, Q425 binding of calcium resulted in a 55,000-fold enhancement in affinity for CD4. X-ray crystallographic analyses of Q425 in the presence of Ca2+, Ba2+, or EDTA revealed an exposed metal-binding site, partially coordinated by five atoms contributed from four antibody complementarity-determining regions. The results suggest that Q425 recognition of CD4 involves direct ligation of antigen by the Q425-held calcium, with calcium binding each ligating atom of CD4 with ≈1.5 kcal/mol of binding energy. This energetic contribution, which is greater than that from a typical protein atom, demonstrates how interfacial metal ligation can play a unique role in antigen recognition. PMID:16195378

Towards advanced biological detection using surface enhanced raman scattering (SERS)-based sensors

NASA Astrophysics Data System (ADS)

Hankus, Mikella E.; Stratis-Cullum, Dimitra N.; Pellegrino, Paul M.

2010-08-01

The Army has a need for an accurate, fast, reliable and robust means to identify and quantify defense related materials. Raman spectroscopy is a form of vibrational spectroscopy that is rapidly becoming a valuable tool for homeland defense applications, as it is well suited for the molecular identification of a variety of compounds, including explosives and chemical and biological hazards. To measure trace levels of these types of materials, surface enhanced Raman scattering (SERS), a specialized form of Raman scattering, can be employed. The SERS enhancements are produced on, or in close proximity to, a nanoscale roughened metal surface and are typically associated with increased local electromagnetic field strengths. However, before application of SERS in the field and in particular to biological and other hazard sensing applications, significant improvements in substrate performance are needed. In this work, we will report the use of several SERS substrate architectures (colloids, film-over-nanospheres (FONs) and commercially available substrates) for detecting and differentiating numerous endospore samples. The variance in spectra as obtained using different sensing architectures will also be discussed. Additionally, the feasibility of using a modified substrate architecture that is tailored with molecular recognition probe system for detecting biological samples will be explored. We will discuss the progress towards an advanced, hybrid molecular recognition with a SERS/Fluorescence nanoprobe system including the optimization, fabrication, and spectroscopic analysis of samples on a commercially available substrate. Additionally, the feasibility of using this single-step switching architecture for hazard material detection will also be explored.

Unique structural modulation of a non-native substrate by cochaperone DnaJ.

PubMed

Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik; Mapa, Koyeli

2013-02-12

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK-DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.

Structural and functional determination of homologs of the Mycobacterium tuberculosisN-acetylglucosamine-6-phosphate deacetylase (NagA).

PubMed

Ahangar, Mohd Syed; Furze, Christopher M; Guy, Collette S; Cooper, Charlotte; Maskew, Kathryn S; Graham, Ben; Cameron, Alexander D; Fullam, Elizabeth

2018-05-04

The Mycobacterium tuberculosis (Mtb) pathogen encodes an N -acetylglucosamine-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of N -acetylglucosamine-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential anti-tubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereo-selective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 Å and 2.0 Å resolutions, respectively. The GlcNAc6P-complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Distinct parietal sites mediate the influences of mood, arousal, and their interaction on human recognition memory.

PubMed

Greene, Ciara M; Flannery, Oliver; Soto, David

2014-12-01

The two dimensions of emotion, mood valence and arousal, have independent effects on recognition memory. At present, however, it is not clear how those effects are reflected in the human brain. Previous research in this area has generally dealt with memory for emotionally valenced or arousing stimuli, but the manner in which interacting mood and arousal states modulate responses in memory substrates remains poorly understood. We investigated memory for emotionally neutral items while independently manipulating mood valence and arousal state by means of music exposure. Four emotional conditions were created: positive mood/high arousal, positive mood/low arousal, negative mood/high arousal, and negative mood/low arousal. We observed distinct effects of mood valence and arousal in parietal substrates of recognition memory. Positive mood increased activity in ventral posterior parietal cortex (PPC) and orbitofrontal cortex, whereas arousal condition modulated activity in dorsal PPC and the posterior cingulate. An interaction between valence and arousal was observed in left ventral PPC, notably in a parietal area distinct from the those identified for the main effects, with a stronger effect of mood on recognition memory responses here under conditions of relative high versus low arousal. We interpreted the PPC activations in terms of the attention-to-memory hypothesis: Increased arousal may lead to increased top-down control of memory, and hence dorsal PPC activation, whereas positive mood valence may result in increased activity in ventral PPC regions associated with bottom-up attention to memory. These findings indicate that distinct parietal sites mediate the influences of mood, arousal, and their interplay during recognition memory.

HIV-1 protease-substrate coevolution in nelfinavir resistance.

PubMed

Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

2014-07-01

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Regulation of Nucleotide Excision Repair by UV-DDB: Prioritization of Damage Recognition to Internucleosomal DNA

PubMed Central

Luch, Andreas; Glas, Andreas; Carell, Thomas; Naegeli, Hanspeter

2011-01-01

How tightly packed chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged DNA-binding protein (UV-DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the nucleosome repeat landscape of chromatin. Therefore, the nucleosomes of human cells have been dissected by micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A ubiquitin ligase activity is necessary to retain the xeroderma pigmentosum group C (XPC) partner at such internucleosomal repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-DDB also adopts a ubiquitin-independent function is evidenced by domain mapping and in situ protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable β-hairpin insertion of XPC into substrate DNA. We conclude that the evolutionary advent of UV-DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic chromatin. PMID:22039351

Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA.

PubMed

Lam, Kwok-Ho; Sikorra, Stefan; Weisemann, Jasmin; Maatsch, Hannah; Perry, Kay; Rummel, Andreas; Binz, Thomas; Jin, Rongsheng

2018-04-23

The extreme toxicity of botulinum neurotoxins (BoNTs) relies on their specific cleavage of SNARE proteins, which eventually leads to muscle paralysis. One newly identified mosaic toxin, BoNT/HA (aka H or FA), cleaves VAMP-2 at a unique position between residues L54 and E55, but the molecular basis underlying VAMP-2-recognition of BoNT/HA remains poorly characterized. Here, we report a ∼2.09 Å resolution crystal structure of the light chain protease domain of BoNT/HA (LC/HA). Structural comparison between LC/HA and LC of BoNT/F1 (LC/F1) reveals distinctive hydrophobic and electrostatic features near the active sites, which may explain their different VAMP-2 cleavage sites. When compared to BoNT/F5 that cleaves VAMP-2 at the same site as BoNT/HA, LC/HA displays higher affinity for VAMP-2, which could be caused by their different surface charge properties surrounding a VAMP-2 exosite-binding cleft. Furthermore, systematic mutagenesis studies on VAMP-2 and structural modeling demonstrate that residues R47 to K59 spanning the cleavage site in VAMP-2 may adopt a novel extended conformation when interacting with LC/HA and LC/F5. Taken together, our structure provides new insights into substrate-recognition of BoNT/HA and paves the way for rational design of small molecule or peptide inhibitors against LC/HA.

Common and Unique Impairments in Facial-Expression Recognition in Pervasive Developmental Disorder-Not Otherwise Specified and Asperger's Disorder

ERIC Educational Resources Information Center

Uono, Shota; Sato, Wataru; Toichi, Motomi

2013-01-01

This study was designed to identify specific difficulties and associated features related to the problems with social interaction experienced by individuals with pervasive developmental disorder-not otherwise specified (PDD-NOS) using an emotion-recognition task. We compared individuals with PDD-NOS or Asperger's disorder (ASP) and typically…

Word Recognition is Affected by the Meaning of Orthographic Neighbours: Evidence from Semantic Decision Tasks

ERIC Educational Resources Information Center

Boot, Inge; Pecher, Diane

2008-01-01

Many models of word recognition predict that neighbours of target words will be activated during word processing. Cascaded models can make the additional prediction that semantic features of those neighbours get activated before the target has been uniquely identified. In two semantic decision tasks neighbours that were congruent (i.e., from the…

Introduction and Overview of the Vicens-Reddy Speech Recognition System.

ERIC Educational Resources Information Center

Kameny, Iris; Ritea, H.

The Vicens-Reddy System is unique in the sense that it approaches the problem of speech recognition as a whole, rather than treating particular aspects of the problems as in previous attempts. For example, where earlier systems treated only segmentation of speech into phoneme groups, or detected phonemes in a given context, the Vicens-Reddy System…

Genetic specificity of face recognition.

PubMed

Shakeshaft, Nicholas G; Plomin, Robert

2015-10-13

Specific cognitive abilities in diverse domains are typically found to be highly heritable and substantially correlated with general cognitive ability (g), both phenotypically and genetically. Recent twin studies have found the ability to memorize and recognize faces to be an exception, being similarly heritable but phenotypically substantially uncorrelated both with g and with general object recognition. However, the genetic relationships between face recognition and other abilities (the extent to which they share a common genetic etiology) cannot be determined from phenotypic associations. In this, to our knowledge, first study of the genetic associations between face recognition and other domains, 2,000 18- and 19-year-old United Kingdom twins completed tests assessing their face recognition, object recognition, and general cognitive abilities. Results confirmed the substantial heritability of face recognition (61%), and multivariate genetic analyses found that most of this genetic influence is unique and not shared with other cognitive abilities.

Genetic specificity of face recognition

PubMed Central

Shakeshaft, Nicholas G.; Plomin, Robert

2015-01-01

Specific cognitive abilities in diverse domains are typically found to be highly heritable and substantially correlated with general cognitive ability (g), both phenotypically and genetically. Recent twin studies have found the ability to memorize and recognize faces to be an exception, being similarly heritable but phenotypically substantially uncorrelated both with g and with general object recognition. However, the genetic relationships between face recognition and other abilities (the extent to which they share a common genetic etiology) cannot be determined from phenotypic associations. In this, to our knowledge, first study of the genetic associations between face recognition and other domains, 2,000 18- and 19-year-old United Kingdom twins completed tests assessing their face recognition, object recognition, and general cognitive abilities. Results confirmed the substantial heritability of face recognition (61%), and multivariate genetic analyses found that most of this genetic influence is unique and not shared with other cognitive abilities. PMID:26417086

Offline Arabic handwriting recognition: a survey.

PubMed

Lorigo, Liana M; Govindaraju, Venu

2006-05-01

The automatic recognition of text on scanned images has enabled many applications such as searching for words in large volumes of documents, automatic sorting of postal mail, and convenient editing of previously printed documents. The domain of handwriting in the Arabic script presents unique technical challenges and has been addressed more recently than other domains. Many different methods have been proposed and applied to various types of images. This paper provides a comprehensive review of these methods. It is the first survey to focus on Arabic handwriting recognition and the first Arabic character recognition survey to provide recognition rates and descriptions of test data for the approaches discussed. It includes background on the field, discussion of the methods, and future research directions.

Effect of Vowel Context on the Recognition of Initial Consonants in Kannada.

PubMed

Kalaiah, Mohan Kumar; Bhat, Jayashree S

2017-09-01

The present study was carried out to investigate the effect of vowel context on the recognition of Kannada consonants in quiet for young adults. A total of 17 young adults with normal hearing in both ears participated in the study. The stimuli included consonant-vowel syllables, spoken by 12 native speakers of Kannada. Consonant recognition task was carried out as a closed-set (fourteen-alternative forced-choice). The present study showed an effect of vowel context on the perception of consonants. Maximum consonant recognition score was obtained in the /o/ vowel context, followed by the /a/ and /u/ vowel contexts, and then the /e/ context. Poorest consonant recognition score was obtained in the vowel context /i/. Vowel context has an effect on the recognition of Kannada consonants, and the vowel effect was unique for Kannada consonants.

Mapping substrate interactions of the human membrane-associated neuraminidase, NEU3, using STD NMR.

PubMed

Albohy, Amgad; Richards, Michele R; Cairo, Christopher W

2015-03-01

Saturation transfer difference (STD) nuclear magnetic resonance (NMR) is a powerful technique which can be used to investigate interactions between proteins and their substrates. The method identifies specific sites of interaction found on a small molecule ligand when in complex with a protein. The ability of STD NMR to provide specific insight into binding interactions in the absence of other structural data is an attractive feature for its use with membrane proteins. We chose to employ STD NMR in our ongoing investigations of the human membrane-associated neuraminidase NEU3 and its interaction with glycolipid substrates (e.g., GM3). In order to identify critical substrate-enzyme interactions, we performed STD NMR with a catalytically inactive form of the enzyme, NEU3(Y370F), containing an N-terminal maltose-binding protein (MBP)-affinity tag. In the absence of crystallographic data on the enzyme, these data represent a critical experimental test of proposed homology models, as well as valuable new structural data. To aid interpretation of the STD NMR data, we compared the results with molecular dynamics (MD) simulations of the enzyme-substrate complexes. We find that the homology model is able to predict essential features of the experimental data, including close contact of the hydrophobic aglycone and the Neu5Ac residue with the enzyme. Additionally, the model and STD NMR data agree on the facial recognition of the galactose and glucose residues of the GM3-analog studied. We conclude that the homology model of NEU3 can be used to predict substrate recognition, but our data indicate that unstructured portions of the NEU3 model may require further refinement. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Neurotransmitter and psychostimulant recognition by the dopamine transporter

PubMed Central

Wang, Kevin H.; Penmatsa, Aravind; Gouaux, Eric

2015-01-01

Na+/Cl−-coupled biogenic amine transporters are the primary targets of therapeutic and abused drugs, ranging from antidepressants to the psychostimulants cocaine and amphetamines, and to their cognate substrates. Here we determine x-ray crystal structures of the Drosophila melanogaster dopamine transporter (dDAT) bound to its substrate dopamine (DA), a substrate analogue 3,4-dichlorophenethylamine, the psychostimulants D-amphetamine, methamphetamine, or to cocaine and cocaine analogues. All ligands bind to the central binding site, located approximately halfway across the membrane bilayer, in close proximity to bound sodium and chloride ions. The central binding site recognizes three chemically distinct classes of ligands via conformational changes that accommodate varying sizes and shapes, thus illustrating molecular principles that distinguish substrates from inhibitors in biogenic amine transporters. PMID:25970245

Kinemage of action - Proposed reaction mechanism of glutamate-1-semialdehyde aminomutase at an atomic level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sorensen, John L., E-mail: John_Sorensen@umanitoba.ca; Stetefeld, Joerg, E-mail: stetefel@cc.umanitoba.ca

2011-10-07

Highlights: {yields} Inhibitors of tetrapyrrole cofactor biosynthesis may be useful antibiotics. {yields} Mechanism of critical enzyme, glutamate-1-semialdehyde aminomutase, is presented. {yields} Unique vitamin B6-dependant enzyme traps intermediate in active site. {yields} Molecular dynamics show that a re-orientation of the substrate is required. -- Abstract: Glutamate-1-semialdehyde aminomutase (GSAM), a key enzyme in tetrapyrrole cofactor biosynthesis, performs a unique transamination on a single substrate. The substrate, glutamate-1-semialdehyde (GSA), undergoes a reaction that exchanges the position of an amine and a carbonyl group to produce 5-aminolevulinic acid (ALA). This transamination reaction is unique in the fact that is does not require an externalmore » cofactor to act as a nitrogen donor or acceptor in this transamination reaction. One of the other remarkable features of the catalytic mechanism is the release free in the enzyme active site of the intermediate 4,5-diaminovaleric acid (DAVA). The action of a gating loop prevents the escape of DAVA from the active site. In a MD simulation approach, using snapshots provided by X-ray crystallography and protein crystal absorption spectrometry data, the individual catalytic steps in this unique intramolecular transamination have been elucidated.« less

Chemical biology-based approaches on fluorescent labeling of proteins in live cells.

PubMed

Jung, Deokho; Min, Kyoungmi; Jung, Juyeon; Jang, Wonhee; Kwon, Youngeun

2013-05-01

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria.

PubMed

Lau, Edward; Wang, Ding; Zhang, Jun; Yu, Hongxiu; Lam, Maggie P Y; Liang, Xiangbo; Zong, Nobel; Kim, Tae-Young; Ping, Peipei

2012-04-27

Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart. To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms. Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography-tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities. The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.

Influenza sensor

DOEpatents

Swanson, Basil I.; Song, Xuedong; Unkefer, Clifford; Silks, III, Louis A.; Schmidt, Jurgen G.

2003-09-30

A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

Influenza Sensor

DOEpatents

Swanson, Basil I.; Song, Xuedong; Unkefer, Clifford; Silks, III, Louis A.; Schmidt, Jurgen G.

2006-03-28

A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

Influenza Sensor

DOEpatents

Swanson, Basil I.; Song, Xuedong; Unkefer, Clifford; Silks, III, Louis A.; Schmidt, Jurgen G.

2005-05-17

A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

Exosites in the substrate specificity of blood coagulation reactions.

PubMed

Bock, P E; Panizzi, P; Verhamme, I M A

2007-07-01

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

Emotion recognition deficits associated with ventromedial prefrontal cortex lesions are improved by gaze manipulation.

PubMed

Wolf, Richard C; Pujara, Maia; Baskaya, Mustafa K; Koenigs, Michael

2016-09-01

Facial emotion recognition is a critical aspect of human communication. Since abnormalities in facial emotion recognition are associated with social and affective impairment in a variety of psychiatric and neurological conditions, identifying the neural substrates and psychological processes underlying facial emotion recognition will help advance basic and translational research on social-affective function. Ventromedial prefrontal cortex (vmPFC) has recently been implicated in deploying visual attention to the eyes of emotional faces, although there is mixed evidence regarding the importance of this brain region for recognition accuracy. In the present study of neurological patients with vmPFC damage, we used an emotion recognition task with morphed facial expressions of varying intensities to determine (1) whether vmPFC is essential for emotion recognition accuracy, and (2) whether instructed attention to the eyes of faces would be sufficient to improve any accuracy deficits. We found that vmPFC lesion patients are impaired, relative to neurologically healthy adults, at recognizing moderate intensity expressions of anger and that recognition accuracy can be improved by providing instructions of where to fixate. These results suggest that vmPFC may be important for the recognition of facial emotion through a role in guiding visual attention to emotionally salient regions of faces. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recycling microcavity optical biosensors.

PubMed

Hunt, Heather K; Armani, Andrea M

2011-04-01

Optical biosensors have tremendous potential for commercial applications in medical diagnostics, environmental monitoring, and food safety evaluation. In these applications, sensor reuse is desirable to reduce costs. To achieve this, harsh, wet chemistry treatments are required to remove surface chemistry from the sensor, typically resulting in reduced sensor performance and increased noise due to recognition moiety and optical transducer degradation. In the present work, we suggest an alternative, dry-chemistry method, based on O₂ plasma treatment. This approach is compatible with typical fabrication of substrate-based optical transducers. This treatment completely removes the recognition moiety, allowing the transducer surface to be refreshed with new recognition elements and thus enabling the sensor to be recycled.

The neural basis of body form and body action agnosia.

PubMed

Moro, Valentina; Urgesi, Cosimo; Pernigo, Simone; Lanteri, Paola; Pazzaglia, Mariella; Aglioti, Salvatore Maria

2008-10-23

Visual analysis of faces and nonfacial body stimuli brings about neural activity in different cortical areas. Moreover, processing body form and body action relies on distinct neural substrates. Although brain lesion studies show specific face processing deficits, neuropsychological evidence for defective recognition of nonfacial body parts is lacking. By combining psychophysics studies with lesion-mapping techniques, we found that lesions of ventromedial, occipitotemporal areas induce face and body recognition deficits while lesions involving extrastriate body area seem causatively associated with impaired recognition of body but not of face and object stimuli. We also found that body form and body action recognition deficits can be double dissociated and are causatively associated with lesions to extrastriate body area and ventral premotor cortex, respectively. Our study reports two category-specific visual deficits, called body form and body action agnosia, and highlights their neural underpinnings.

Toward a Comprehensive System of Personnel Development in Deafblind Education

ERIC Educational Resources Information Center

Parker, Amy T.; Nelson, Catherine

2016-01-01

Students who are deafblind are a unique population with unique needs for learning, communication, and environmental access. Two roles have been identified as important to their education: teacher of the deafblind and intervener. However, these roles are not officially recognized in most states. Because of this lack of recognition and the low…

Extending enzyme molecular recognition with an expanded amino acid alphabet

PubMed Central

Windle, Claire L.; Simmons, Katie J.; Ault, James R.; Trinh, Chi H.; Nelson, Adam

2017-01-01

Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties. PMID:28196894

Complementary and partially complementary DNA duplexes tethered to a functionalized substrate: a molecular dynamics approach to biosensing.

PubMed

Monti, Susanna; Cacelli, Ivo; Ferretti, Alessandro; Prampolini, Giacomo; Barone, Vincenzo

2011-07-21

Molecular dynamics simulations (90 ns) of different DNA complexes attached to a functionalized substrate in solution were performed in order to clarify the behavior of mismatched DNA sequences captured by a tethered DNA probe (biochip). Examination of the trajectories revealed that the substrate influence and a series of cooperative events, including recognition, reorientation and reorganization of the bases, could induce the formation of stable duplexes having non-canonical arrangements. Major adjustment of the structures was observed when the mutated base was located in the end region of the chain close to the surface. This journal is © the Owner Societies 2011

Structure-Guided Functional Characterization of DUF1460 Reveals a Highly Specific NlpC/P60 Amidase Family

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Patin, Delphine; ...

2014-11-20

GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a novel, highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo-structure at 1.15 Å resolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predictedmore » in silico based on structural and bioinformatics data, and were subsequently characterized experimentally. Ultimately, further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines a new amidase family.« less

Structure-Guided Functional Characterization of DUF1460 Reveals a Highly Specific NlpC/P60 Amidase Family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Patin, Delphine

GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a novel, highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo-structure at 1.15 Å resolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predictedmore » in silico based on structural and bioinformatics data, and were subsequently characterized experimentally. Ultimately, further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines a new amidase family.« less

Structure-guided functional characterization of DUF1460 reveals a highly specific NlpC/P60 amidase family.

PubMed

Xu, Qingping; Mengin-Lecreulx, Dominique; Patin, Delphine; Grant, Joanna C; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W; Godzik, Adam; Lesley, Scott A; Elsliger, Marc-André; Deacon, Ashley M; Wilson, Ian A

2014-12-02

GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo structure at 1.15 Å resolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predicted in silico based on structural and bioinformatics data, and subsequently were characterized experimentally. Further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines another amidase family. Copyright © 2014 Elsevier Ltd. All rights reserved.

Role of the α clamp in the protein translocation mechanism of anthrax toxin

PubMed Central

Brown, Michael J.; Thoren, Katie L.; Krantz, Bryan A.

2015-01-01

Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins—protective antigen (PA), lethal factor (LF), and edema factor (EF). Upon endocytosis into host cells, acidification of the endosome induces PA to form a membrane-inserted channel, which unfolds LF and EF and translocates them into the host cytosol. Translocation is driven by the proton motive force, comprised of the chemical potential, the proton-gradient (ΔpH), and the membrane potential (ΔΨ). A crystal structure of the lethal toxin core complex revealed an “α clamp” structure that binds to substrate helices nonspecifically. Here we test the hypothesis that through the recognition of unfolding helical structure the α clamp can accelerate the rate of translocation. We produced a synthetic PA mutant in which an α helix was crosslinked into the α clamp to block its function. This synthetic construct impairs translocation by raising a yet uncharacterized translocation barrier shown to be much less force dependent than the known unfolding barrier. We also report that the α clamp more stably binds substrates that can form helices than those, such as polyproline, that cannot. Hence the α clamp recognizes substrates by a general shape-complementarity mechanism. Substrates that are incapable of forming compact secondary structure (due to the introduction of a polyproline track) are severely deficient for translocation. Therefore, the α clamp and its recognition of helical structure in the translocating substrate play key roles in the molecular mechanism of protein translocation. PMID:26344833

Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal R.; Swaminathan S.; Burley, S. K.

2012-01-01

The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyzes conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyzes phosphorylation of all four 2-ketopentose sugars with comparable k{sub cat} values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptidemore » chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified {sup 447}{und GG}LPQ{und K}{sup 452} as the ATP-binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the side chains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.« less

Six characteristics of effective structured reporting and the inevitable integration with speech recognition.

PubMed

Liu, David; Zucherman, Mark; Tulloss, William B

2006-03-01

The reporting of radiological images is undergoing dramatic changes due to the introduction of two new technologies: structured reporting and speech recognition. Each technology has its own unique advantages. The highly organized content of structured reporting facilitates data mining and billing, whereas speech recognition offers a natural succession from the traditional dictation-transcription process. This article clarifies the distinction between the process and outcome of structured reporting, describes fundamental requirements for any effective structured reporting system, and describes the potential development of a novel, easy-to-use, customizable structured reporting system that incorporates speech recognition. This system should have all the advantages derived from structured reporting, accommodate a wide variety of user needs, and incorporate speech recognition as a natural component and extension of the overall reporting process.

Wearable Spiral Passive Electromagnetic Sensor (SPES) glove for sign language recognition of alphabet letters and numbers: a preliminary study

NASA Astrophysics Data System (ADS)

Iervolino, Onorio; Meo, Michele

2017-04-01

Sign language is a method of communication for deaf-mute people with articulated gestures and postures of hands and fingers to represent alphabet letters or complete words. Recognizing gestures is a difficult task, due to intrapersonal and interpersonal variations in performing them. This paper investigates the use of Spiral Passive Electromagnetic Sensor (SPES) as a motion recognition tool. An instrumented glove integrated with wearable multi-SPES sensors was developed to encode data and provide a unique response for each hand gesture. The device can be used for recognition of gestures; motion control and well-defined gesture sets such as sign languages. Each specific gesture was associated to a unique sensor response. The gloves encode data regarding the gesture directly in the frequency spectrum response of the SPES. The absence of chip or complex electronic circuit make the gloves light and comfortable to wear. Results showed encouraging data to use SPES in wearable applications.

Preparation of high-strength nanometer scale twinned coating and foil

DOEpatents

Zhang, Xinghang [Los Alamos, NM; Misra, Amit [Los Alamos, NM; Nastasi, Michael A [Santa Fe, NM; Hoagland, Richard G [Santa Fe, NM

2006-07-18

Very high strength single phase stainless steel coating has been prepared by magnetron sputtering onto a substrate. The coating has a unique microstructure of nanometer spaced twins that are parallel to each other and to the substrate surface. For cases where the coating and substrate do not bind strongly, the coating can be peeled off to provide foil.

Silicon nanomembranes as a means to evaluate stress evolution in deposited thin films

Treesearch

Anna M. Clausen; Deborah M. Paskiewicz; Alireza Sadeghirad; Joseph Jakes; Donald E. Savage; Donald S. Stone; Feng Liu; Max G. Lagally

2014-01-01

Thin-film deposition on ultra-thin substrates poses unique challenges because of the potential for a dynamic response to the film stress during deposition. While theoretical studies have investigated film stress related changes in bulk substrates, little has been done to learn how stress might evolve in a film growing on a compliant substrate. We use silicon...

Layered Metal Nanoparticle Structures on Electrodes for Sensing, Switchable Controlled Uptake/Release, and Photo-electrochemical Applications.

PubMed

Tel-Vered, Ran; Kahn, Jason S; Willner, Itamar

2016-01-06

Layered metal nanoparticle (NP) assemblies provide highly porous and conductive composites of unique electrical and optical (plasmonic) properties. Two methods to construct layered metal NP matrices are described, and these include the layer-by-layer deposition of NPs, or the electropolymerization of monolayer-functionalized NPs, specifically thioaniline-modified metal NPs. The layered NP composites are used as sensing matrices through the use of electrochemistry or surface plasmon resonance (SPR) as transduction signals. The crosslinking of the metal NP composites with molecular receptors, or the imprinting of molecular recognition sites into the electropolymerized NP matrices lead to selective and chiroselective sensing interfaces. Furthermore, the electrosynthesis of redox-active, imprinted, bis-aniline bridged Au NP composites yields electrochemically triggered "sponges" for the switchable uptake and release of electron-acceptor substrates, and results in conductive surfaces of electrochemically controlled wettability. Also, photosensitizer-relay-crosslinked Au NP composites, or electrochemically polymerized layered semiconductor quantum dot/metal NP matrices on electrodes, are demonstrated as functional nanostructures for photoelectrochemical applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrolytic lesions of dorsal CA3 impair episodic-like memory in rats.

PubMed

Li, Jay-Shake; Chao, Yuen-Shin

2008-02-01

Episodic memory is the ability to recollect one's past experiences occurring in an unique spatial and temporal context. In non-human animals, it is expressed in the ability to combine "what", "where" and "when" factors to form an integrated memory system. During the search for its neural substrates, the hippocampus has attracted a lot of attentions. Yet, it is not yet possible to induce a pure episodic-like memory deficit in animal studies without being confounded by impairments in the spatial cognition. Here, we present a lesion study evidencing direct links between the hippocampus CA3 region and the episodic-like memory in rats. In a spontaneous object exploration task, lesioned rats showed no interaction between the temporal and spatial elements in their memory associated with the objects. In separate tests carried out subsequently, the same animals still expressed abilities to process spatial, temporal, and object recognition memory. In conclusions, our results support the idea that the hippocampus CA3 has a particular status in the neural mechanism of the episodic-like memory system. It is responsible for combining information from different modules of cognitive processes.

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP.

PubMed

Bickford, Justin S; Nick, Harry S

2013-12-01

Isoprenoid lipid carriers are essential in protein glycosylation and bacterial cell envelope biosynthesis. The enzymes involved in their metabolism (synthases, kinases and phosphatases) are therefore critical to cell viability. In this review, we focus on two broad groups of isoprenoid pyrophosphate phosphatases. One group, containing phosphatidic acid phosphatase motifs, includes the eukaryotic dolichyl pyrophosphate phosphatases and proposed recycling bacterial undecaprenol pyrophosphate phosphatases, PgpB, YbjB and YeiU/LpxT. The second group comprises the bacterial undecaprenol pyrophosphate phosphatase, BacA/UppP, responsible for initial formation of undecaprenyl phosphate, which we predict contains a tyrosine phosphate phosphatase motif resembling that of the tumour suppressor, phosphatase and tensin homologue (PTEN). Based on protein sequence alignments across species and 2D structure predictions, we propose catalytic and lipid recognition motifs unique to BacA/UppP enzymes. The verification of our proposed active-site residues would provide new strategies for the development of substrate-specific inhibitors which mimic both the lipid and pyrophosphate moieties, leading to the development of novel antimicrobial agents.

Bio-recognitive photonics of a DNA-guided organic semiconductor

PubMed Central

Back, Seung Hyuk; Park, Jin Hyuk; Cui, Chunzhi; Ahn, Dong June

2016-01-01

Incorporation of duplex DNA with higher molecular weights has attracted attention for a new opportunity towards a better organic light-emitting diode (OLED) capability. However, biological recognition by OLED materials is yet to be addressed. In this study, specific oligomeric DNA–DNA recognition is successfully achieved by tri (8-hydroxyquinoline) aluminium (Alq3), an organic semiconductor. Alq3 rods crystallized with guidance from single-strand DNA molecules show, strikingly, a unique distribution of the DNA molecules with a shape of an ‘inverted' hourglass. The crystal's luminescent intensity is enhanced by 1.6-fold upon recognition of the perfect-matched target DNA sequence, but not in the case of a single-base mismatched one. The DNA–DNA recognition forming double-helix structure is identified to occur only in the rod's outer periphery. This study opens up new opportunities of Alq3, one of the most widely used OLED materials, enabling biological recognition. PMID:26725969

Cost-sensitive learning for emotion robust speaker recognition.

PubMed

Li, Dongdong; Yang, Yingchun; Dai, Weihui

2014-01-01

In the field of information security, voice is one of the most important parts in biometrics. Especially, with the development of voice communication through the Internet or telephone system, huge voice data resources are accessed. In speaker recognition, voiceprint can be applied as the unique password for the user to prove his/her identity. However, speech with various emotions can cause an unacceptably high error rate and aggravate the performance of speaker recognition system. This paper deals with this problem by introducing a cost-sensitive learning technology to reweight the probability of test affective utterances in the pitch envelop level, which can enhance the robustness in emotion-dependent speaker recognition effectively. Based on that technology, a new architecture of recognition system as well as its components is proposed in this paper. The experiment conducted on the Mandarin Affective Speech Corpus shows that an improvement of 8% identification rate over the traditional speaker recognition is achieved.

Cost-Sensitive Learning for Emotion Robust Speaker Recognition

PubMed Central

Li, Dongdong; Yang, Yingchun

2014-01-01

In the field of information security, voice is one of the most important parts in biometrics. Especially, with the development of voice communication through the Internet or telephone system, huge voice data resources are accessed. In speaker recognition, voiceprint can be applied as the unique password for the user to prove his/her identity. However, speech with various emotions can cause an unacceptably high error rate and aggravate the performance of speaker recognition system. This paper deals with this problem by introducing a cost-sensitive learning technology to reweight the probability of test affective utterances in the pitch envelop level, which can enhance the robustness in emotion-dependent speaker recognition effectively. Based on that technology, a new architecture of recognition system as well as its components is proposed in this paper. The experiment conducted on the Mandarin Affective Speech Corpus shows that an improvement of 8% identification rate over the traditional speaker recognition is achieved. PMID:24999492

Bio-recognitive photonics of a DNA-guided organic semiconductor.

PubMed

Back, Seung Hyuk; Park, Jin Hyuk; Cui, Chunzhi; Ahn, Dong June

2016-01-04

Incorporation of duplex DNA with higher molecular weights has attracted attention for a new opportunity towards a better organic light-emitting diode (OLED) capability. However, biological recognition by OLED materials is yet to be addressed. In this study, specific oligomeric DNA-DNA recognition is successfully achieved by tri (8-hydroxyquinoline) aluminium (Alq3), an organic semiconductor. Alq3 rods crystallized with guidance from single-strand DNA molecules show, strikingly, a unique distribution of the DNA molecules with a shape of an 'inverted' hourglass. The crystal's luminescent intensity is enhanced by 1.6-fold upon recognition of the perfect-matched target DNA sequence, but not in the case of a single-base mismatched one. The DNA-DNA recognition forming double-helix structure is identified to occur only in the rod's outer periphery. This study opens up new opportunities of Alq3, one of the most widely used OLED materials, enabling biological recognition.

Bio-recognitive photonics of a DNA-guided organic semiconductor

NASA Astrophysics Data System (ADS)

Back, Seung Hyuk; Park, Jin Hyuk; Cui, Chunzhi; Ahn, Dong June

2016-01-01

Incorporation of duplex DNA with higher molecular weights has attracted attention for a new opportunity towards a better organic light-emitting diode (OLED) capability. However, biological recognition by OLED materials is yet to be addressed. In this study, specific oligomeric DNA-DNA recognition is successfully achieved by tri (8-hydroxyquinoline) aluminium (Alq3), an organic semiconductor. Alq3 rods crystallized with guidance from single-strand DNA molecules show, strikingly, a unique distribution of the DNA molecules with a shape of an `inverted' hourglass. The crystal's luminescent intensity is enhanced by 1.6-fold upon recognition of the perfect-matched target DNA sequence, but not in the case of a single-base mismatched one. The DNA-DNA recognition forming double-helix structure is identified to occur only in the rod's outer periphery. This study opens up new opportunities of Alq3, one of the most widely used OLED materials, enabling biological recognition.

Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway. PMID:25941524

Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

Facial recognition performance of female inmates as a result of sexual assault history.

PubMed

Islam-Zwart, Kayleen A; Heath, Nicole M; Vik, Peter W

2005-06-01

This study examined the effect of sexual assault history on facial recognition performance. Gender of facial stimuli and posttraumatic stress disorder (PTSD) symptoms also were expected to influence performance. Fifty-six female inmates completed an interview and the Wechsler Memory Scale-Third Edition Faces I and Faces II subtests (Wechsler, 1997). Women with a sexual assault exhibited better immediate and delayed facial recognition skills than those with no assault history. There were no differences in performance based on the gender of faces or PTSD diagnosis. Immediate facial recognition was correlated with report of PTSD symptoms. Findings provide greater insight into women's reactions to, and the uniqueness of, the trauma of sexual victimization.

Discrete Neural Correlates for the Recognition of Negative Emotions: Insights from Frontotemporal Dementia

PubMed Central

Kumfor, Fiona; Irish, Muireann; Hodges, John R.; Piguet, Olivier

2013-01-01

Patients with frontotemporal dementia have pervasive changes in emotion recognition and social cognition, yet the neural changes underlying these emotion processing deficits remain unclear. The multimodal system model of emotion proposes that basic emotions are dependent on distinct brain regions, which undergo significant pathological changes in frontotemporal dementia. As such, this syndrome may provide important insight into the impact of neural network degeneration upon the innate ability to recognise emotions. This study used voxel-based morphometry to identify discrete neural correlates involved in the recognition of basic emotions (anger, disgust, fear, sadness, surprise and happiness) in frontotemporal dementia. Forty frontotemporal dementia patients (18 behavioural-variant, 11 semantic dementia, 11 progressive nonfluent aphasia) and 27 healthy controls were tested on two facial emotion recognition tasks: The Ekman 60 and Ekman Caricatures. Although each frontotemporal dementia group showed impaired recognition of negative emotions, distinct associations between emotion-specific task performance and changes in grey matter intensity emerged. Fear recognition was associated with the right amygdala; disgust recognition with the left insula; anger recognition with the left middle and superior temporal gyrus; and sadness recognition with the left subcallosal cingulate, indicating that discrete neural substrates are necessary for emotion recognition in frontotemporal dementia. The erosion of emotion-specific neural networks in neurodegenerative disorders may produce distinct profiles of performance that are relevant to understanding the neurobiological basis of emotion processing. PMID:23805313

Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies.

PubMed

Ye, Yuxin; Saburi, Wataru; Odaka, Rei; Kato, Koji; Sakurai, Naofumi; Komoda, Keisuke; Nishimoto, Mamoru; Kitaoka, Motomitsu; Mori, Haruhide; Yao, Min

2016-03-01

In Ruminococcus albus, 4-O-β-D-mannosyl-D-glucose phosphorylase (RaMP1) and β-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze β-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-β-D-mannosyl-d-glucose and RaMP2 with/without β-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding. © 2016 Federation of European Biochemical Societies.

Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

PubMed Central

Martin, Teresa A.; Herman, Christine T.; Limpoco, Francis T.; Michael, Madeline C.; Potts, Gregory K.; Bailey, Ryan C.

2014-01-01

Methods for the generation of substrates presenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retention of function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor-ligand interactions. This molecularly general photochemical patterning method is an enabling tool that will allow the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces. PMID:21793535

Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study†

PubMed Central

Peng, Dungeng; Satterlee, James D.; Ma, Li-Hua; Dallas, Jerry L.; Smith, Kevin M.; Zhang, Xuhong; Sato, Michihiko; La Mar, Gerd N.

2011-01-01

Heme oxygenase, HO, from the pathogenic bacterium N. meningitidis, NmHO, which secures host iron, shares many properties with mammalian HOs, but also exhibits some key differences. The crystal structure appears more compact and the crystal-undetected C-terminus interacts with substrate in solution. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D 1H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~102 increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed. PMID:21870860

Facial emotion recognition in patients with focal and diffuse axonal injury.

PubMed

Yassin, Walid; Callahan, Brandy L; Ubukata, Shiho; Sugihara, Genichi; Murai, Toshiya; Ueda, Keita

2017-01-01

Facial emotion recognition impairment has been well documented in patients with traumatic brain injury. Studies exploring the neural substrates involved in such deficits have implicated specific grey matter structures (e.g. orbitofrontal regions), as well as diffuse white matter damage. Our study aims to clarify whether different types of injuries (i.e. focal vs. diffuse) will lead to different types of impairments on facial emotion recognition tasks, as no study has directly compared these patients. The present study examined performance and response patterns on a facial emotion recognition task in 14 participants with diffuse axonal injury (DAI), 14 with focal injury (FI) and 22 healthy controls. We found that, overall, participants with FI and DAI performed more poorly than controls on the facial emotion recognition task. Further, we observed comparable emotion recognition performance in participants with FI and DAI, despite differences in the nature and distribution of their lesions. However, the rating response pattern between the patient groups was different. This is the first study to show that pure DAI, without gross focal lesions, can independently lead to facial emotion recognition deficits and that rating patterns differ depending on the type and location of trauma.

Toward a unified model of face and object recognition in the human visual system

PubMed Central

Wallis, Guy

2013-01-01

Our understanding of the mechanisms and neural substrates underlying visual recognition has made considerable progress over the past 30 years. During this period, accumulating evidence has led many scientists to conclude that objects and faces are recognised in fundamentally distinct ways, and in fundamentally distinct cortical areas. In the psychological literature, in particular, this dissociation has led to a palpable disconnect between theories of how we process and represent the two classes of object. This paper follows a trend in part of the recognition literature to try to reconcile what we know about these two forms of recognition by considering the effects of learning. Taking a widely accepted, self-organizing model of object recognition, this paper explains how such a system is affected by repeated exposure to specific stimulus classes. In so doing, it explains how many aspects of recognition generally regarded as unusual to faces (holistic processing, configural processing, sensitivity to inversion, the other-race effect, the prototype effect, etc.) are emergent properties of category-specific learning within such a system. Overall, the paper describes how a single model of recognition learning can and does produce the seemingly very different types of representation associated with faces and objects. PMID:23966963

“A room full of strangers every day”: The psychosocial impact of developmental prosopagnosia on children and their families

PubMed Central

Dalrymple, Kirsten A.; Fletcher, Kimberley; Corrow, Sherryse; Nair, Roshan das; Barton, Jason J. S.; Yonas, Albert; Duchaine, Brad

2014-01-01

Objective Individuals with developmental prosopagnosia (‘face blindness’) have severe face recognition difficulties due to a failure to develop the necessary visual mechanisms for recognizing faces. These difficulties occur in the absence of brain damage and despite normal low-level vision and intellect. Adults with developmental prosopagnosia report serious personal and emotional consequences from their inability to recognize faces, but little is known about the psychosocial consequences in childhood. Given the importance of face recognition in daily life, and the potential for unique social consequences of impaired face recognition in childhood, we sought to evaluate the impact of developmental prosopagnosia on children and their families. Methods We conducted semi-structured interviews with 8 children with developmental prosopagnosia and their parents. A battery of face recognition tests was used to confirm the face recognition impairment reported by the parents of each child. We used thematic analysis to develop common themes among the psychosocial experiences of the children and their parents. Results Three themes were developed from the child reports: 1) awareness of their difficulties, 2) coping strategies, such as using non-facial cues to identify others, and 3) social implications, such as discomfort in, and avoidance of, social situations. These themes were paralleled by the parent reports and highlight the unique social and practical challenges associated with childhood developmental prosopagnosia. Conclusion Our findings indicate a need for increased awareness and treatment of developmental prosopagnosia to help these children manage their face recognition difficulties and to promote their social and emotional wellbeing. PMID:25077856

Molecular simulations bring new insights into flavonoid/quercetinase interaction modes.

PubMed

Fiorucci, Sébastien; Golebiowski, Jérôme; Cabrol-Bass, Daniel; Antonczak, Serge

2007-06-01

Molecular dynamics simulations, using the AMBER force field, were performed to study Quercetin 2,3-Dioxygenase enzyme (Quercetinase or 2,3QD). We have analyzed the structural modifications of the active site and of the linker region between the native enzyme and the enzyme-substrate complex. New structural informations, such as an allosteric effect in the presence of the substrate, as well as description of the enzyme-substrate interactions and values of binding free energies were brought out. All these results confirm the idea that the linker encloses the substrate in the active site and also enlighten the recognition role of the substrate B-ring by the enzyme. Moreover, a specific interaction scheme has been proposed to explain the relative degradation rate of various flavonoid compounds under the oxygenolysis reaction catalyzed by the Quercetin 2,3-Dioxygenase enzyme. 2007 Wiley-Liss, Inc.

Understanding Substrate Selectivity of Human UDP-glucuronosyltransferases through QSAR modeling and analysis of homologous enzymes

PubMed Central

Dong, Dong; Ako, Roland; Hu, Ming; Wu, Baojian

2015-01-01

The UDP-glucuronosyltransferase (UGT) enzyme catalyzes the glucuronidation reaction which is a major metabolic and detoxification pathway in humans. Understanding the mechanisms for substrate recognition by UGT assumes great importance in an attempt to predict its contribution to xenobiotic/drug disposition in vivo. Spurred on by this interest, 2D/3D-quantitative structure activity relationships (QSAR) and pharmacophore models have been established in the absence of a complete mammalian UGT crystal structure. This review discusses the recent progress in modeling human UGT substrates including those with multiple sites of glucuronidation. A better understanding of UGT active site contributing to substrate selectivity (and regioselectivity) from the homologous enzymes (i.e., plant and bacterial UGTs, all belong to family 1 of glycosyltransferase (GT1)) is also highlighted, as these enzymes share a common catalytic mechanism and/or overlapping substrate selectivity. PMID:22385482

Analyte-Responsive Hydrogels: Intelligent Materials for Biosensing and Drug Delivery.

PubMed

Culver, Heidi R; Clegg, John R; Peppas, Nicholas A

2017-02-21

Nature has mastered the art of molecular recognition. For example, using synergistic non-covalent interactions, proteins can distinguish between molecules and bind a partner with incredible affinity and specificity. Scientists have developed, and continue to develop, techniques to investigate and better understand molecular recognition. As a consequence, analyte-responsive hydrogels that mimic these recognitive processes have emerged as a class of intelligent materials. These materials are unique not only in the type of analyte to which they respond but also in how molecular recognition is achieved and how the hydrogel responds to the analyte. Traditional intelligent hydrogels can respond to environmental cues such as pH, temperature, and ionic strength. The functional monomers used to make these hydrogels can be varied to achieve responsive behavior. For analyte-responsive hydrogels, molecular recognition can also be achieved by incorporating biomolecules with inherent molecular recognition properties (e.g., nucleic acids, peptides, enzymes, etc.) into the polymer network. Furthermore, in addition to typical swelling/syneresis responses, these materials exhibit unique responsive behaviors, such as gel assembly or disassembly, upon interaction with the target analyte. With the diverse tools available for molecular recognition and the ability to generate unique responsive behaviors, analyte-responsive hydrogels have found great utility in a wide range of applications. In this Account, we discuss strategies for making four different classes of analyte-responsive hydrogels, specifically, non-imprinted, molecularly imprinted, biomolecule-containing, and enzymatically responsive hydrogels. Then we explore how these materials have been incorporated into sensors and drug delivery systems, highlighting examples that demonstrate the versatility of these materials. For example, in addition to the molecular recognition properties of analyte-responsive hydrogels, the physicochemical changes that are induced upon analyte binding can be exploited to generate a detectable signal for sensing applications. As research in this area has grown, a number of creative approaches for improving the selectivity and sensitivity (i.e., detection limit) of these sensors have emerged. For applications in drug delivery systems, therapeutic release can be triggered by competitive molecular interactions or physicochemical changes in the network. Additionally, including degradable units within the network can enable sustained and responsive therapeutic release. Several exciting examples exploiting the analyte-responsive behavior of hydrogels for the treatment of cancer, diabetes, and irritable bowel syndrome are discussed in detail. We expect that creative and combinatorial approaches used in the design of analyte-responsive hydrogels will continue to yield materials with great potential in the fields of sensing and drug delivery.

Human body as a set of biometric features identified by means of optoelectronics

NASA Astrophysics Data System (ADS)

Podbielska, Halina; Bauer, Joanna

2005-09-01

Human body posses many unique, singular features that are impossible to copy or forge. Nowadays, to establish and to ensure the public security requires specially designed devices and systems. Biometrics is a field of science and technology, exploiting human body characteristics for people recognition. It identifies the most characteristic and unique ones in order to design and construct systems capable to recognize people. In this paper some overview is given, presenting the achievements in biometrics. The verification and identification process is explained, along with the way of evaluation of biometric recognition systems. The most frequently human biometrics used in practice are shortly presented, including fingerprints, facial imaging (including thermal characteristic), hand geometry and iris patterns.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

NASA Astrophysics Data System (ADS)

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-01

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.

PubMed

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-30

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD + -dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some "loose-binding" substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Graphene transport mediated by micropatterned substrates

NASA Astrophysics Data System (ADS)

Hinnefeld, J. Henry; Gill, Stephen T.; Mason, Nadya

2018-04-01

Engineered substrates offer a promising avenue towards graphene devices having tunable properties. In particular, topographically patterned substrates can expose unique behavior due to their ability to induce local variations in strain and electrostatic doping. However, to explore the range of possible science and applications, it is important to create topographic substrates that both have tunable features and are suitable for transport measurements. In this letter, we describe the fabrication of tunable, topographically patterned substrates suitable for transport measurements. We report both optical and transport measurements of graphene devices fabricated on these substrates and demonstrate the characteristic strain and local doping behavior induced by the topographic features.

Molecular recognition of 7-(2-octadecyloxycarbonylethyl)guanine to cytidine at the air/water interface and LB film studied by Fourier transform infrared spectroscopy

NASA Astrophysics Data System (ADS)

Miao, Wangen; Luo, Xuzhong; Liang, Yingqiu

2003-03-01

Monolayer behavior of a nucleolipid amphiphile, 7-(2-octadecyloxycarbonylethyl)guanine (ODCG), on aqueous cytidine solution was investigated by means of surface-molecular area ( π- A) isotherms. It indicates that molecular recognition by hydrogen bonding is present between ODCG monolayer and the cytidine in subphase. The Fourier transform infrared (FTIR) transmission spectroscopic result indicates that the cytidine molecules in the subphase can be transferred onto solid substrates by Langmuir-Blodgett (LB) technique as a result of the formation of Watson-Crick base-pairing at the air/water interface. Investigation by rotating polarized FTIR transmission also suggests that the headgroup recognition of this amphiphile to the dissolved cytidine influence the orientation of the tailchains.

Cross spectral, active and passive approach to face recognition for improved performance

NASA Astrophysics Data System (ADS)

Grudzien, A.; Kowalski, M.; Szustakowski, M.

2017-08-01

Biometrics is a technique for automatic recognition of a person based on physiological or behavior characteristics. Since the characteristics used are unique, biometrics can create a direct link between a person and identity, based on variety of characteristics. The human face is one of the most important biometric modalities for automatic authentication. The most popular method of face recognition which relies on processing of visual information seems to be imperfect. Thermal infrared imagery may be a promising alternative or complement to visible range imaging due to its several reasons. This paper presents an approach of combining both methods.

New endophytic Toxicocladosporium species from cacti in Brazil, and description of Neocladosporium gen. nov.

PubMed

Bezerra, Jadson D P; Sandoval-Denis, Marcelo; Paiva, Laura M; Silva, Gladstone A; Groenewald, Johannes Z; Souza-Motta, Cristina M; Crous, Pedro W

2017-06-01

Brazil harbours a unique ecosystem, the Caatinga, which belongs to the tropical dry forest biome. This region has an important diversity of organisms, and recently several new fungal species have been described from different hosts and substrates within it. During a survey of fungal endophyte diversity from cacti in this forest, we isolated cladosporium-like fungi that were subjected to morphological and multigene phylogenetic analyses including actA , ITS, LSU, rpb2 and tub2 gene sequences. Based on these analyses we identified two new species belonging to the genus Toxicocladosporium , described here as T. cacti and T. immaculatum spp. nov., isolated from Pilosocereus gounellei subsp. gounellei and Melocactus zehntneri , respectively. To improve the species recognition and assess species diversity in Toxicocladosporium we studied all ex-type strains of the genus, for which actA , rpb2 and tub2 barcodes were also generated. After phylogenetic reconstruction using five loci, we differentiated 13 species in the genus. Toxicocladosporium velox and T. chlamydosporum are synonymized based on their phylogenetic position and limited number of unique nucleotide differences. Six strains previously assigned to T. leucadendri , including the ex-type strain (CBS 131317) of that species, were found to belong to an undescribed genus here named as Neocladosporium gen. nov., with N. leucadendri comb. nov. as type species. Furthermore, this study proposes the actA , ITS, rpb2 and tub2 as main phylogenetic loci to recognise Toxicocladosporium species.

Unveiling NIR Aza-Boron-Dipyrromethene (BODIPY) Dyes as Raman Probes: Surface-Enhanced Raman Scattering (SERS)-Guided Selective Detection and Imaging of Human Cancer Cells.

PubMed

Adarsh, Nagappanpillai; Ramya, Adukkadan N; Maiti, Kaustabh Kumar; Ramaiah, Danaboyina

2017-10-12

The development of new Raman reporters has attracted immense attention in diagnostic research based on surface enhanced Raman scattering (SERS) techniques, which is a well established method for ultrasensitive detection through molecular fingerprinting and imaging. Herein, for the first time, we report the unique and efficient Raman active features of the selected aza-BODIPY dyes 1-6. These distinctive attributes could be extended at the molecular level to allow detection through SERS upon adsorption onto nano-roughened gold surface. Among the newly revealed Raman reporters, the amino substituted derivative 4 showed high signal intensity at very low concentrations (ca. 0.4 μm for 4-Au). Interestingly, an efficient nanoprobe has been constructed by using gold nanoparticles as SERS substrate, and 4 as the Raman reporter (4-Au@PEG), which unexpectedly showed efficient recognition of three human cancer cells (lung: A549, cervical: HeLa, Fibrosarcoma: HT-1080) without any specific surface marker. We observed well reflected and resolved Raman mapping and characteristic signature peaks whereas, such recognition was not observed in normal fibroblast (3T3L1) cells. To confirm these findings, a SERS nanoprobe was conjugated with a specific tumour targeting marker, EGFR (Epidermal Growth Factor Receptor), a well known targeted agent for Human Fibrosarcoma (HT1080). This nanoprobe efficiently targeted the surface marker of HT1080 cells, threreby demonstrating its use as an ultrasensitive Raman probe for detection and targeted imaging, leaving normal cells unaffected. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Polybasic Region of the Polysialyltransferase ST8Sia-IV Binds Directly to the Neural Cell Adhesion Molecule, NCAM.

PubMed

Bhide, Gaurang P; Prehna, Gerd; Ramirez, Benjamin E; Colley, Karen J

2017-03-14

Polysialic acid (polySia) is a unique post-translational modification found on a small set of mammalian glycoproteins. Composed of long chains of α2,8-linked sialic acid, this large, negatively charged polymer attenuates protein and cell adhesion and modulates signaling mediated by its carriers and proteins that interact with these carriers. PolySia is crucial for the proper development of the nervous system and is upregulated during tissue regeneration and in highly invasive cancers. Our laboratory has previously shown that the neural cell adhesion molecule, NCAM, has an acidic surface patch in its first fibronectin type III repeat (FN1) that is critical for the polysialylation of N-glycans on the adjacent immunoglobulin domain (Ig5). We have also identified a polysialyltransferase (polyST) polybasic region (PBR) that may mediate substrate recognition. However, a direct interaction between the NCAM FN1 acidic patch and the polyST PBR has yet to be demonstrated. Here, we have probed this interaction using isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. We observe direct and specific binding between FN1 and the PBR peptide that is dependent upon acidic residues in FN1 and basic residues of the PBR. NMR titration experiments verified the role of the FN1 acidic patch in the recognition of the PBR and suggest a conformational change of the Ig5-FN1 linker region following binding of the PBR to the acidic patch. Finally, mutation of residues identified by NMR titration experiments impacts NCAM polysialylation, supporting their mechanistic role in protein-specific polysialylation.

Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.

PubMed

Ma, Xianyue; Cline, Kenneth

2013-03-01

Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

Development of First-Graders' Word Reading Skills: For Whom Can Dynamic Assessment Tell Us More?

PubMed

Cho, Eunsoo; Compton, Donald L; Gilbert, Jennifer K; Steacy, Laura M; Collins, Alyson A; Lindström, Esther R

2017-01-01

Dynamic assessment (DA) of word reading measures learning potential for early reading development by documenting the amount of assistance needed to learn how to read words with unfamiliar orthography. We examined the additive value of DA for predicting first-grade decoding and word recognition development while controlling for autoregressive effects. Additionally, we examined whether predictive validity of DA would be higher for students who have poor phonological awareness skills. First-grade students (n = 105) were assessed on measures of word reading, phonological awareness, rapid automatized naming, and DA in the fall and again assessed on word reading measures in the spring. A series of planned, moderated multiple regression analyses indicated that DA made a significant and unique contribution in predicting word recognition development above and beyond the autoregressor, particularly for students with poor phonological awareness skills. For these students, DA explained 3.5% of the unique variance in end-of-first-grade word recognition that was not attributable to autoregressive effect. Results suggest that DA provides an important source of individual differences in the development of word recognition skills that cannot be fully captured by merely assessing the present level of reading skills through traditional static assessment, particularly for students at risk for developing reading disabilities. © Hammill Institute on Disabilities 2015.

The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

PubMed

Roth, Braden M; Ishimaru, Daniella; Hennig, Mirko

2013-09-13

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

The Core Microprocessor Component DiGeorge Syndrome Critical Region 8 (DGCR8) Is a Nonspecific RNA-binding Protein*

PubMed Central

Roth, Braden M.; Ishimaru, Daniella; Hennig, Mirko

2013-01-01

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins. PMID:23893406

The “gating” residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition

PubMed Central

Milczek, Erika M.; Binda, Claudia; Rovida, Stefano; Mattevi, Andrea; Edmondson, Dale E.

2011-01-01

Summary The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å3 volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å3 (entrance cavity) and ~400 Å3 (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared to WT enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100–103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine KM but unaltered kcat values. The altered KM values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to WT enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provides insights into specific reversible inhibitor design for these membrane-bound enzymes. PMID:21978362

Recognition and Resistance in TEM [superscript beta]-Lactamase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiaojun; Minasov, George; Blazquez, Jesus

Developing antimicrobials that are less likely to engender resistance has become an important design criterion as more and more drugs fall victim to resistance mutations. One hypothesis is that the more closely an inhibitor resembles a substrate, the more difficult it will be to develop resistant mutations that can at once disfavor the inhibitor and still recognize the substrate. To investigate this hypothesis, 10 transition-state analogues, of greater or lesser similarity to substrates, were tested for inhibition of TEM-1 beta-lactamase, the most widespread resistance enzyme to penicillin antibiotics. The inhibitors were also tested against four characteristic mutant enzymes: TEM-30, TEM-32,more » TEM-52, and TEM-64. The inhibitor most similar to the substrate, compound 10, was the most potent inhibitor of the WT enzyme, with a K(i) value of 64 nM. Conversely, compound 10 was the most susceptible to the TEM-30 (R244S) mutant, for which inhibition dropped by over 100-fold. The other inhibitors were relatively impervious to the TEM-30 mutant enzyme. To understand recognition and resistance to these transition-state analogues, the structures of four of these inhibitors in complex with TEM-1 were determined by X-ray crystallography. These structures suggest a structural basis for distinguishing inhibitors that mimic the acylation transition state and those that mimic the deacylation transition state; they also suggest how TEM-30 reduces the affinity of compound 10. In cell culture, this inhibitor reversed the resistance of bacteria to ampicillin, reducing minimum inhibitory concentrations of this penicillin by between 4- and 64-fold, depending on the strain of bacteria. Notwithstanding this activity, the resistance of TEM-30, which is already extant in the clinic, suggests that there can be resistance liabilities with substrate-based design.« less

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

PubMed

Mpakali, Anastasia; Giastas, Petros; Mathioudakis, Nikolas; Mavridis, Irene M; Saridakis, Emmanuel; Stratikos, Efstratios

2015-10-23

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

PubMed Central

Simone, Domenico; Bay, Denice C.; Leach, Thorin; Turner, Raymond J.

2013-01-01

Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial classes and its specialization may be driven by the substrates it transports and the environment of its host. PMID:24236045

Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

PubMed

Hu, Xiao-Qian; Guo, Peng-Chao; Ma, Jin-Di; Li, Wei-Fang

2013-11-01

The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases.

PubMed

Lohman, Jeremy R; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

PubMed Central

Lohman, Jeremy R.; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

2015-01-01

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs. PMID:26420866

Kinome signaling through regulated protein-protein interactions in normal and cancer cells.

PubMed

Pawson, Tony; Kofler, Michael

2009-04-01

The flow of molecular information through normal and oncogenic signaling pathways frequently depends on protein phosphorylation, mediated by specific kinases, and the selective binding of the resulting phosphorylation sites to interaction domains present on downstream targets. This physical and functional interplay of catalytic and interaction domains can be clearly seen in cytoplasmic tyrosine kinases such as Src, Abl, Fes, and ZAP-70. Although the kinase and SH2 domains of these proteins possess similar intrinsic properties of phosphorylating tyrosine residues or binding phosphotyrosine sites, they also undergo intramolecular interactions when linked together, in a fashion that varies from protein to protein. These cooperative interactions can have diverse effects on substrate recognition and kinase activity, and provide a variety of mechanisms to link the stimulation of catalytic activity to substrate recognition. Taken together, these data have suggested how protein kinases, and the signaling pathways in which they are embedded, can evolve complex properties through the stepwise linkage of domains within single polypeptides or multi-protein assemblies.

Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

T Wang; K Heran Darwin; H Li

2011-12-31

Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Ourmore » work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.« less

Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, T.; Li, H.; Darwin, K. H.

2010-11-01

Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Ourmore » work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.« less

Complete abolition of reading and writing ability with a third ventricle colloid cyst: implications for surgical intervention and proposed neural substrates of visual recognition and visual imaging ability.

PubMed

Barker, Lynne Ann; Morton, Nicholas; Romanowski, Charles A J; Gosden, Kevin

2013-10-24

We report a rare case of a patient unable to read (alexic) and write (agraphic) after a mild head injury. He had preserved speech and comprehension, could spell aloud, identify words spelt aloud and copy letter features. He was unable to visualise letters but showed no problems with digits. Neuropsychological testing revealed general visual memory, processing speed and imaging deficits. Imaging data revealed an 8 mm colloid cyst of the third ventricle that splayed the fornix. Little is known about functions mediated by fornical connectivity, but this region is thought to contribute to memory recall. Other regions thought to mediate letter recognition and letter imagery, visual word form area and visual pathways were intact. We remediated reading and writing by multimodal letter retraining. The study raises issues about the neural substrates of reading, role of fornical tracts to selective memory in the absence of other pathology, and effective remediation strategies for selective functional deficits.

Nucleosome Recognition by the Piccolo NuA4 Histone Acetyltransferase Complex†

PubMed Central

Berndsen, Christopher E.; Selleck, William; McBryant, Steven J.; Hansen, Jeffrey C.; Tan, Song; Demi, John M.

2007-01-01

The mechanisms by which multisubunit histone acetyltransferase (HAT) complexes recognize and perform efficient acetylation on nucleosome substrates are largely unknown. Here, we use a variety of biochemical approaches and compare histone-based substrates of increasing complexity to determine the critical components of nucleosome recognition by the MOZ, Ybf2/Sas3, Sas2, Tip60 family HAT complex, Piccolo NuA4 (picNuA4). We find the histone tails to be dispensable for binding to both nucleosomes and free histones and that the H2A, H3, and H2B tails do not influence the ability of picNuA4 to tetra-acetylate the H4 tail within the nucleosome. Most notably, we discovered that the histone-fold domain (HFD) regions of histones, particularly residues 21–52 of H4, are critical for tight binding and efficient tail acetylation. Presented evidence suggests that picNuA4 recognizes the open surface of the nucleosome on which the HFD of H4 is located. This binding mechanism serves to direct substrate access to the tails of H4 and H2A and allows the enzyme to be “tethered”, thereby increasing the effective concentration of the histone tail and permitting successive cycles of H4 tail acetylation. PMID:17274630

Chlorella virus DNA ligase: nick recognition and mutational analysis.

PubMed

Sriskanda, V; Shuman, S

1998-01-15

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.

Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

PubMed Central

Mobley, E M; Pan, T

1999-01-01

Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate. PMID:10518624

Discovery of a Xylooligosaccharide Oxidase from Myceliophthora thermophila C1.

PubMed

Ferrari, Alessandro R; Rozeboom, Henriëtte J; Dobruchowska, Justyna M; van Leeuwen, Sander S; Vugts, Aniek S C; Koetsier, Martijn J; Visser, Jaap; Fraaije, Marco W

2016-11-04

By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.

PubMed

Ong, Jennifer L; Loakes, David; Jaroslawski, Szymon; Too, Kathleen; Holliger, Philipp

2006-08-18

DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes.

Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

PubMed

Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi

2015-01-01

In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for identifying ubiquitination sites. The proposed method presents a practical means of preliminary analysis and greatly diminishes the total number of potential targets required for further experimental confirmation. This method may help unravel their mechanisms and roles in E3 recognition and ubiquitin-mediated protein degradation.

Dioxygen in Polyoxometalate Mediated Reactions.

PubMed

Weinstock, Ira A; Schreiber, Roy E; Neumann, Ronny

2018-03-14

In this review article, we consider the use of molecular oxygen in reactions mediated by polyoxometalates. Polyoxometalates are anionic metal oxide clusters of a variety of structures that are soluble in liquid phases and therefore amenable to homogeneous catalytic transformations. Often, they are active for electron transfer oxidations of a myriad of substrates and upon reduction can be reoxidized by molecular oxygen. For example, the phosphovanadomolybdate, H 5 PV 2 Mo 10 O 40 , can oxidize Pd(0) thereby enabling aerobic reactions catalyzed by Pd and H 5 PV 2 Mo 10 O 40 . In a similar vein, polyoxometalates can stabilize metal nanoparticles, leading to additional transformations. Furthermore, electron transfer oxidation of other substrates such as halides and sulfur-containing compounds is possible. More uniquely, H 5 PV 2 Mo 10 O 40 and its analogues can mediate electron transfer-oxygen transfer reactions where oxygen atoms are transferred from the polyoxometalate to the substrate. This unique property has enabled correspondingly unique transformations involving carbon-carbon, carbon-hydrogen, and carbon-metal bond activation. The pathway for the reoxidation of vanadomolybdates with O 2 appears to be an inner-sphere reaction, but the oxidation of one-electron reduced polyoxotungstates has been shown through intensive research to be an outer-sphere reaction. Beyond electron transfer and electron transfer-oxygen transfer aerobic transformations, there a few examples of apparent dioxygenase activity where both oxygen atoms are donated to a substrate.

Assessing collective affect recognition via the Emotional Aperture Measure.

PubMed

Sanchez-Burks, Jeffrey; Bartel, Caroline A; Rees, Laura; Huy, Quy

2016-01-01

Curiosity about collective affect is undergoing a revival in many fields. This literature, tracing back to Le Bon's seminal work on crowd psychology, has established the veracity of collective affect and demonstrated its influence on a wide range of group dynamics. More recently, an interest in the perception of collective affect has emerged, revealing a need for a methodological approach for assessing collective emotion recognition to complement measures of individual emotion recognition. This article addresses this need by introducing the Emotional Aperture Measure (EAM). Three studies provide evidence that collective affect recognition requires a processing style distinct from individual emotion recognition and establishes the validity and reliability of the EAM. A sample of working managers further shows how the EAM provides unique insights into how individuals interact with collectives. We discuss how the EAM can advance several lines of research on collective affect.

Finger vein recognition based on finger crease location

NASA Astrophysics Data System (ADS)

Lu, Zhiying; Ding, Shumeng; Yin, Jing

2016-07-01

Finger vein recognition technology has significant advantages over other methods in terms of accuracy, uniqueness, and stability, and it has wide promising applications in the field of biometric recognition. We propose using finger creases to locate and extract an object region. Then we use linear fitting to overcome the problem of finger rotation in the plane. The method of modular adaptive histogram equalization (MAHE) is presented to enhance image contrast and reduce computational cost. To extract the finger vein features, we use a fusion method, which can obtain clear and distinguishable vein patterns under different conditions. We used the Hausdorff average distance algorithm to examine the recognition performance of the system. The experimental results demonstrate that MAHE can better balance the recognition accuracy and the expenditure of time compared with three other methods. Our resulting equal error rate throughout the total procedure was 3.268% in a database of 153 finger vein images.

SAR target recognition using behaviour library of different shapes in different incidence angles and polarisations

NASA Astrophysics Data System (ADS)

Fallahpour, Mojtaba Behzad; Dehghani, Hamid; Jabbar Rashidi, Ali; Sheikhi, Abbas

2018-05-01

Target recognition is one of the most important issues in the interpretation of the synthetic aperture radar (SAR) images. Modelling, analysis, and recognition of the effects of influential parameters in the SAR can provide a better understanding of the SAR imaging systems, and therefore facilitates the interpretation of the produced images. Influential parameters in SAR images can be divided into five general categories of radar, radar platform, channel, imaging region, and processing section, each of which has different physical, structural, hardware, and software sub-parameters with clear roles in the finally formed images. In this paper, for the first time, a behaviour library that includes the effects of polarisation, incidence angle, and shape of targets, as radar and imaging region sub-parameters, in the SAR images are extracted. This library shows that the created pattern for each of cylindrical, conical, and cubic shapes is unique, and due to their unique properties these types of shapes can be recognised in the SAR images. This capability is applied to data acquired with the Canadian RADARSAT1 satellite.

Specific Impairments in the Recognition of Emotional Facial Expressions in Parkinson’s Disease

PubMed Central

Clark, Uraina S.; Neargarder, Sandy; Cronin-Golomb, Alice

2008-01-01

Studies investigating the ability to recognize emotional facial expressions in non-demented individuals with Parkinson’s disease (PD) have yielded equivocal findings. A possible reason for this variability may lie in the confounding of emotion recognition with cognitive task requirements, a confound arising from the lack of a control condition using non-emotional stimuli. The present study examined emotional facial expression recognition abilities in 20 non-demented patients with PD and 23 control participants relative to their performances on a non-emotional landscape categorization test with comparable task requirements. We found that PD participants were normal on the control task but exhibited selective impairments in the recognition of facial emotion, specifically for anger (driven by those with right hemisphere pathology) and surprise (driven by those with left hemisphere pathology), even when controlling for depression level. Male but not female PD participants further displayed specific deficits in the recognition of fearful expressions. We suggest that the neural substrates that may subserve these impairments include the ventral striatum, amygdala, and prefrontal cortices. Finally, we observed that in PD participants, deficiencies in facial emotion recognition correlated with higher levels of interpersonal distress, which calls attention to the significant psychosocial impact that facial emotion recognition impairments may have on individuals with PD. PMID:18485422

Diversity in recognition of glycans by F-type lectins and galectins: molecular, structural, and biophysical aspects

PubMed Central

Vasta, Gerardo R.; Ahmed, Hafiz; Bianchet, Mario A.; Fernández-Robledo, José A.; Amzel, L. Mario

2013-01-01

Although lectins are “hard-wired” in the germline, the presence of tandemly arrayed carbohydrate recognition domains (CRDs), of chimeric structures displaying distinct CRDs, of polymorphic genes resulting in multiple isoforms, and in some cases, of a considerable recognition plasticity of their carbohydrate binding sites, significantly expand the lectin ligand-recognition spectrum and lectin functional diversification. Analysis of structural/functional aspects of galectins and F-lectins—the most recently identified lectin family characterized by a unique CRD sequence motif (a distinctive structural fold) and nominal specificity for l-Fuc—has led to a greater understanding of self/nonself recognition by proteins with tandemly arrayed CRDs. For lectins with a single CRD, however, recognition of self and nonself glycans can only be rationalized in terms of protein oligomerization and ligand clustering and presentation. Spatial and temporal changes in lectin expression, secretion, and local concentrations in extracellular microenvironments, as well as structural diversity and spatial display of their carbohydrate ligands on the host or microbial cell surface, are suggestive of a dynamic interplay of their recognition and effector functions in development and immunity. PMID:22973821

Catalytic Function and Substrate Specificity of the Papain-Like Protease Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus

PubMed Central

Báez-Santos, Yahira M.; Mielech, Anna M.; Deng, Xufang; Baker, Susan

2014-01-01

ABSTRACT The papain-like protease (PLpro) domain from the deadly Middle East respiratory syndrome coronavirus (MERS-CoV) was overexpressed and purified. MERS-CoV PLpro constructs with and without the putative ubiquitin-like (UBL) domain at the N terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncovered significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin, and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have 8-fold and 3,500-fold higher catalytic efficiencies for hydrolysis of ISG15-AMC than for hydrolysis of the Ub-AMC and Z-RLRGG-AMC substrates, respectively. A similar trend was observed for SARS-CoV PLpro, although it was much more efficient than MERS-CoV PLpro toward ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains at similar rates and with similar debranching patterns, producing monoubiquitin species. However, SARS-CoV PLpro much preferred K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produced di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of the MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition. IMPORTANCE Unlocking the secrets of how coronavirus (CoV) papain-like proteases (PLpros) perform their multifunctional roles during viral replication entails a complete mechanistic understanding of their substrate recognition and enzymatic activities. We show that the PLpro domains from the MERS and SARS coronaviruses can recognize and process the same substrates, but with different catalytic efficiencies. The differences in substrate recognition between these closely related PLpros suggest that neither enzyme can be used as a generalized model to explain the kinetic behavior of all CoV PLpros. As a consequence, decoding the mechanisms of PLpro-mediated antagonism of the host innate immune response and the development of anti-CoV PLpro enzyme inhibitors will be a challenging undertaking. The results from this study provide valuable information for understanding how MERS-CoV PLpro-mediated antagonism of the host innate immune response is orchestrated, as well as insight into the design of inhibitors against MERS-CoV PLpro. PMID:25142582

Molecular recognition of 7-(2-octadecyloxycarbonylethyl)guanine to cytidine at the air/water interface and LB film studied by Fourier transform infrared spectroscopy.

PubMed

Miao, Wangen; Luo, Xuzhong; Liang, Yingqiu

2003-03-15

Monolayer behavior of a nucleolipid amphiphile, 7-(2-octadecyloxycarbonylethyl)guanine (ODCG), on aqueous cytidine solution was investigated by means of surface-molecular area (pi-A) isotherms. It indicates that molecular recognition by hydrogen bonding is present between ODCG monolayer and the cytidine in subphase. The Fourier transform infrared (FTIR) transmission spectroscopic result indicates that the cytidine molecules in the subphase can be transferred onto solid substrates by Langmuir-Blodgett (LB) technique as a result of the formation of Watson-Crick base-pairing at the air/water interface. Investigation by rotating polarized FTIR transmission also suggests that the headgroup recognition of this amphiphile to the dissolved cytidine influence the orientation of the tailchains. Copyright 2002 Elsevier Science B.V.

Deficits in facial affect recognition among antisocial populations: a meta-analysis.

PubMed

Marsh, Abigail A; Blair, R J R

2008-01-01

Individuals with disorders marked by antisocial behavior frequently show deficits in recognizing displays of facial affect. Antisociality may be associated with specific deficits in identifying fearful expressions, which would implicate dysfunction in neural structures that subserve fearful expression processing. A meta-analysis of 20 studies was conducted to assess: (a) if antisocial populations show any consistent deficits in recognizing six emotional expressions; (b) beyond any generalized impairment, whether specific fear recognition deficits are apparent; and (c) if deficits in fear recognition are a function of task difficulty. Results show a robust link between antisocial behavior and specific deficits in recognizing fearful expressions. This impairment cannot be attributed solely to task difficulty. These results suggest dysfunction among antisocial individuals in specified neural substrates, namely the amygdala, involved in processing fearful facial affect.

Quantitative determination of low-Z elements in single atmospheric particles on boron substrates by automated scanning electron microscopy-energy-dispersive X-ray spectrometry.

PubMed

Choël, Marie; Deboudt, Karine; Osán, János; Flament, Pascal; Van Grieken, René

2005-09-01

Atmospheric aerosols consist of a complex heterogeneous mixture of particles. Single-particle analysis techniques are known to provide unique information on the size-resolved chemical composition of aerosols. A scanning electron microscope (SEM) combined with a thin-window energy-dispersive X-ray (EDX) detector enables the morphological and elemental analysis of single particles down to 0.1 microm with a detection limit of 1-10 wt %, low-Z elements included. To obtain data statistically representative of the air masses sampled, a computer-controlled procedure can be implemented in order to run hundreds of single-particle analyses (typically 1000-2000) automatically in a relatively short period of time (generally 4-8 h, depending on the setup and on the particle loading). However, automated particle analysis by SEM-EDX raises two practical challenges: the accuracy of the particle recognition and the reliability of the quantitative analysis, especially for micrometer-sized particles with low atomic number contents. Since low-Z analysis is hampered by the use of traditional polycarbonate membranes, an alternate choice of substrate is a prerequisite. In this work, boron is being studied as a promising material for particle microanalysis. As EDX is generally said to probe a volume of approximately 1 microm3, geometry effects arise from the finite size of microparticles. These particle geometry effects must be corrected by means of a robust concentration calculation procedure. Conventional quantitative methods developed for bulk samples generate elemental concentrations considerably in error when applied to microparticles. A new methodology for particle microanalysis, combining the use of boron as the substrate material and a reverse Monte Carlo quantitative program, was tested on standard particles ranging from 0.25 to 10 microm. We demonstrate that the quantitative determination of low-Z elements in microparticles is achievable and that highly accurate results can be obtained using the automatic data processing described here compared to conventional methods.

Jump into a New Fold—A Homology Based Model for the ABCG2/BCRP Multidrug Transporter

PubMed Central

László, Laura; Sarkadi, Balázs

2016-01-01

ABCG2/BCRP is a membrane protein, involved in xenobiotic and endobiotic transport in key pharmacological barriers and drug metabolizing organs, in the protection of stem cells, and in multidrug resistance of cancer. Pharmacogenetic studies implicated the role of ABCG2 in response to widely used medicines and anticancer agents, as well as in gout. Its Q141K variant exhibits decreased functional expression thus increased drug accumulation and decreased urate secretion. Still, there has been no reliable molecular model available for this protein, as the published structures of other ABC transporters could not be properly fitted to the ABCG2 topology and experimental data. The recently published high resolution structure of a close homologue, the ABCG5-ABCG8 heterodimer, revealed a new ABC transporter fold, unique for ABCG proteins. Here we present a structural model of the ABCG2 homodimer based on this fold and detail the experimental results supporting this model. In order to describe the effect of mutations on structure and dynamics, and characterize substrate recognition and cholesterol regulation we performed molecular dynamics simulations using full length ABCG2 protein embedded in a membrane bilayer and in silico docking simulations. Our results show that in the Q141K variant the introduced positive charge diminishes the interaction between the nucleotide binding and transmembrane domains and the R482G variation alters the orientation of transmembrane helices. Moreover, the R482 position, which plays a role the substrate specificity of the transporter, is located in one of the substrate binding pockets identified by the in silico docking calculations. In summary, the ABCG2 model and in silico simulations presented here may have significant impact on understanding drug distribution and toxicity, as well as drug development against cancer chemotherapy resistance or gout. PMID:27741279

Wet-surface–enhanced ellipsometric contrast microscopy identifies slime as a major adhesion factor during bacterial surface motility

PubMed Central

Ducret, Adrien; Valignat, Marie-Pierre; Mouhamar, Fabrice; Mignot, Tâm; Theodoly, Olivier

2012-01-01

In biology, the extracellular matrix (ECM) promotes both cell adhesion and specific recognition, which is essential for central developmental processes in both eukaryotes and prokaryotes. However, live studies of the dynamic interactions between cells and the ECM, for example during motility, have been greatly impaired by imaging limitations: mostly the ability to observe the ECM at high resolution in absence of specific staining by live microscopy. To solve this problem, we developed a unique technique, wet-surface enhanced ellipsometry contrast (Wet-SEEC), which magnifies the contrast of transparent organic materials deposited on a substrate (called Wet-surf) with exquisite sensitivity. We show that Wet-SEEC allows both the observation of unprocessed nanofilms as low as 0.2 nm thick and their accurate 3D topographic reconstructions, directly by standard light microscopy. We next used Wet-SEEC to image slime secretion, a poorly defined property of many prokaryotic and eukaryotic organisms that move across solid surfaces in absence of obvious extracellular appendages (gliding). Using combined Wet-SEEC and fluorescent-staining experiments, we observed slime deposition by gliding Myxococcus xanthus cells at unprecedented resolution. Altogether, the results revealed that in this bacterium, slime associates preferentially with the outermost components of the motility machinery and promotes its adhesion to the substrate on the ventral side of the cell. Strikingly, analogous roles have been proposed for the extracellular proteoglycans of gliding diatoms and apicomplexa, suggesting that slime deposition is a general means for gliding organisms to adhere and move over surfaces. PMID:22665761

Common and unique gray matter correlates of episodic memory dysfunction in frontotemporal dementia and Alzheimer's disease.

PubMed

Irish, Muireann; Piguet, Olivier; Hodges, John R; Hornberger, Michael

2014-04-01

Conflicting evidence exists regarding the integrity of episodic memory in the behavioral variant of frontotemporal dementia (bvFTD). Recent converging evidence suggests that episodic memory in progressive cases of bvFTD is compromised to the same extent as in Alzheimer's disease (AD). The underlying neural substrates of these episodic memory deficits, however, likely differ contingent on dementia type. In this study we sought to elucidate the neural substrates of episodic memory performance, across recall and recognition tasks, in both patient groups using voxel-based morphometry (VBM) analyses. We predicted that episodic memory dysfunction would be apparent in both patient groups but would relate to divergent patterns of neural atrophy specific to each dementia type. We assessed episodic memory, across verbal and visual domains, in 19 bvFTD, 18 AD patients, and 19 age- and education-matched controls. Behaviorally, patient groups were indistinguishable for immediate and delayed recall, across verbal and visual domains. Whole-brain VBM analyses revealed regions commonly implicated in episodic retrieval across groups, namely the right temporal pole, right frontal lobe, left paracingulate gyrus, and right anterior hippocampus. Divergent neural networks specific to each group were also identified. Whereas a widespread network including posterior regions such as the posterior cingulate cortex, parietal and occipital cortices was exclusively implicated in AD, the frontal and anterior temporal lobes underpinned the episodic memory deficits in bvFTD. Our results point to distinct neural changes underlying episodic memory decline specific to each dementia syndrome. Copyright © 2013 Wiley Periodicals, Inc.

A complex solution to a sexual dilemma.

PubMed

Kuwabara, Patricia E

2007-07-01

The C. elegans male sex-determining protein, FEM-1, has been identified as a substrate recognition subunit of a Cullin-2 ubiquitin ligase complex. This complex controls the level of TRA-1A, a Ci/Gli homolog and master regulator of sex determination, by ubiquitin-mediated proteolysis.

Molecular tips for scanning tunneling microscopy: intermolecular electron tunneling for single-molecule recognition and electronics.

PubMed

Nishino, Tomoaki

2014-01-01

This paper reviews the development of molecular tips for scanning tunneling microscopy (STM). Molecular tips offer many advantages: first is their ability to perform chemically selective imaging because of chemical interactions between the sample and the molecular tip, thus improving a major drawback of conventional STM. Rational design of the molecular tip allows sophisticated chemical recognition; e.g., chiral recognition and selective visualization of atomic defects in carbon nanotubes. Another advantage is that they provide a unique method to quantify electron transfer between single molecules. Understanding such electron transfer is mandatory for the realization of molecular electronics.

Structural Basis for Substrate Recognition by the Ankyrin Repeat Domain of Human DHHC17 Palmitoyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verardi, Raffaello; Kim, Jin-Sik; Ghirlando, Rodolfo

DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment ofmore » Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most physiologically relevant substrates.« less

Insights into the Specificity of Lysine Acetyltransferases

DOE PAGES

Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; ...

2014-11-07

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. In this paper, we report the structure of a GNATmore » in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Finally, our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.« less

Computer-aided active-site-directed modeling of the Herpes Simplex Virus 1 and human thymidine kinase

NASA Astrophysics Data System (ADS)

Folkers, Gerd; Trumpp-Kallmeyer, Susanne; Gutbrod, Oliver; Krickl, Sabine; Fetzer, Jürgen; Keil, Günther M.

1991-10-01

Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core β-sheet structure, connected by loops and α-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site into Asn. First investigations reveal that the enzymatic activity of the mutant protein is destroyed.

A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.

PubMed

Wood, Sarah E; Sinsinbar, Gaurav; Gudlur, Sushanth; Nallani, Madhavan; Huang, Che-Fan; Liedberg, Bo; Mrksich, Milan

2017-12-22

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k cat /K m value of 6.1×10 6  L mol -1  s -1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

PubMed

Akparov, Valery; Timofeev, Vladimir; Khaliullin, Ilyas; Švedas, Vytas; Kuranova, Inna

2018-03-01

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.

Biochemical analysis and the preliminary crystallographic characterization of D-tagatose 3-epimerase from Rhodobacter sphaeroides.

PubMed

Qi, Zhengliang; Zhu, Zhangliang; Wang, Jian-Wen; Li, Songtao; Guo, Qianqian; Xu, Panpan; Lu, Fuping; Qin, Hui-Min

2017-11-09

D-Tagatose 3-epimerase epimerizes D-fructose to yield D-psicose, which is a rare sugar that exists in small quantities in nature and is difficult to synthesize chemically. We aim to explore potential industrial biocatalysts for commercial-scale manufacture of this rare sugar. A D-tagatose 3-epimerase from Rhodobacter sphaeroides (RsDTE) has recently been identified as a D-tagatose 3-epimerase that can epimerize D-fructose to yield D-psicose with a high conversion rate. The purified RsDTE by Ni-affinity chromatography, ionic exchange chromatography and gel filtration forms a tetramer in solution. The maximal activity was in Tris-HCl buffer pH 8.5, and the optimal temperature was at 35 °C. The product, D-psicose, was confirmed using HPLC and NMR. Crystals of RsDTE were obtained using crystal kits and further refined under crystallization conditions such as 10% PEG 8000,0.1 M HEPES pH 7.5, and 8% ethylene glycol at 20 °C using the sitting-drop vapor diffusion method. The RsDTE homology model showed that it possessed the characteristic TIM-barrel fold. Four residues, Glu156, Asp189, Gln215 and Glu250, forms a hydrogen bond network with the active Mn(II) for the hydride transfer reaction. These residues may constitute the catalytic tetrad of RsDTE. The residues around O1, O2 and O3 of the substrates were conserved. However, the binding-site residues are different at O4, O5 and O6. Arg118 formed the unique hydrogen bond with O4 of D-fructose which indicates RsDTE's preference of D-fructose more than any other family enzymes. RsDTE possesses a different metal-binding site. Arg118, forming unique hydrogen bond with O4 of D-fructose, regulates the substrate recognition. The research on D-tagatose 3-epimerase or D-psicose 3-epimerase enzymes attracts enormous commercial interest and would be widely used for rare sugar production in the future.

STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C.

PubMed

Gaßmeyer, Sarah Katharina; Yoshikawa, Hiroyuki; Enoki, Junichi; Hülsemann, Nadine; Stoll, Raphael; Miyamoto, Kenji; Kourist, Robert

2015-06-23

Structure-guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation-transfer-difference NMR (STD-NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineered unique elastic modes at a BaTiO 3/2x1-Ge(001) interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumah, D. P.; Dogan, M.; Ngai, J. H.

Here, the strong interaction at an interface between a substrate and thin film leads to epitaxy and provides a means of inducing structural changes in the epitaxial film. These induced material phases often exhibit technologically relevant electronic, magnetic, and functional properties. The 2×1 surface of a Ge(001) substrate applies a unique type of epitaxial constraint on thin films of the perovskite oxide BaTiO 3 where a change in bonding and symmetry at the interface leads to a non-bulk-like crystal structure of the BaTiO 3. While the complex crystal structure is predicted using first-principles theory, it is further shown that themore » details of the structure are a consequence of hidden phases found in the bulk elastic response of the BaTiO 3 induced by the symmetry of forces exerted by the germanium substrate.« less

Engineered Unique Elastic Modes at a BaTiO 3 / ( 2 × 1 ) - Ge ( 001 ) Interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumah, D. P.; Dogan, M.; Ngai, J. H.

The strong interaction at an interface between a substrate and thin film leads to epitaxy and provides a means of inducing structural changes in the epitaxial film. These induced material phases often exhibit technologically relevant electronic, magnetic, and functional properties. The 2×1 surface of a Ge(001) substrate applies a unique type of epitaxial constraint on thin films of the perovskite oxide BaTiO3 where a change in bonding and symmetry at the interface leads to a non-bulk-like crystal structure of the BaTiO3. While the complex crystal structure is predicted using first-principles theory, it is further shown that the details of themore » structure are a consequence of hidden phases found in the bulk elastic response of the BaTiO3 induced by the symmetry of forces exerted by the germanium substrate.« less

Engineered unique elastic modes at a BaTiO 3/2x1-Ge(001) interface

DOE PAGES

Kumah, D. P.; Dogan, M.; Ngai, J. H.; ...

2016-03-07

Here, the strong interaction at an interface between a substrate and thin film leads to epitaxy and provides a means of inducing structural changes in the epitaxial film. These induced material phases often exhibit technologically relevant electronic, magnetic, and functional properties. The 2×1 surface of a Ge(001) substrate applies a unique type of epitaxial constraint on thin films of the perovskite oxide BaTiO 3 where a change in bonding and symmetry at the interface leads to a non-bulk-like crystal structure of the BaTiO 3. While the complex crystal structure is predicted using first-principles theory, it is further shown that themore » details of the structure are a consequence of hidden phases found in the bulk elastic response of the BaTiO 3 induced by the symmetry of forces exerted by the germanium substrate.« less

Common folds and transport mechanisms of secondary active transporters.

PubMed

Shi, Yigong

2013-01-01

Secondary active transporters exploit the electrochemical potential of solutes to shuttle specific substrate molecules across biological membranes, usually against their concentration gradient. Transporters of different functional families with little sequence similarity have repeatedly been found to exhibit similar folds, exemplified by the MFS, LeuT, and NhaA folds. Observations of multiple conformational states of the same transporter, represented by the LeuT superfamily members Mhp1, AdiC, vSGLT, and LeuT, led to proposals that structural changes are associated with substrate binding and transport. Despite recent biochemical and structural advances, our understanding of substrate recognition and energy coupling is rather preliminary. This review focuses on the common folds and shared transport mechanisms of secondary active transporters. Available structural information generally supports the alternating access model for substrate transport, with variations and extensions made by emerging structural, biochemical, and computational evidence.

Elastase-like Activity Is Dominant to Chymotrypsin-like Activity in 20S Proteasome's β5 Catalytic Subunit.

PubMed

Bensinger, Dennis; Neumann, Theresa; Scholz, Christoph; Voss, Constantin; Knorr, Sabine; Kuckelkorn, Ulrike; Hamacher, Kay; Kloetzel, Peter-Michael; Schmidt, Boris

2016-07-15

The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotes with several key catalytic cores. Targeting the β5 subunit with small-molecule inhibitors is an established therapeutic strategy for hematologic cancers. Herein, we report a mouse-trap-like conformational change that influences molecular recognition depending on the substitution pattern of a bound ligand. Variation of the size of P1 residues from the highly β5-selective proteasome inhibitor BSc2118 allows for discrimination between inhibitory strength and substrate conversion. We found that increasing molecular size strengthens inhibition, whereas decreasing P1 size accelerates substrate conversion. Evaluation of substrate hydrolysis after silencing of β5 activity reveals significant residual activity for large residues exclusively. Thus, classification of the β5 subunit as chymotrypsin-like and the use of the standard tyrosine-containing substrate should be reconsidered.

Molecular mechanism of substrate recognition and transport by the AtSWEET13 sugar transporter.

PubMed

Han, Lei; Zhu, Yongping; Liu, Min; Zhou, Ye; Lu, Guangyuan; Lan, Lan; Wang, Xianping; Zhao, Yongfang; Zhang, Xuejun C

2017-09-19

Sugar Will Eventually be Exported Transporters (SWEETs) are recently identified sugar transporters that can discriminate and transport di- or monosaccharides across a membrane following the concentration gradient. SWEETs play key roles in plant biological processes, such as pollen nutrition, nectar secretion, seed filling, and phloem loading. SWEET13 from Arabidopsis thaliana (AtSWEET13) is an important sucrose transporter in pollen development. Here, we report the 2.8-Å resolution crystal structure of AtSWEET13 in the inward-facing conformation with a substrate analog, 2'-deoxycytidine 5'-monophosphate, bound in the central cavity. In addition, based on the results of an in-cell transport activity assay and single-molecule Förster resonance energy transfer analysis, we suggest a mechanism for substrate selectivity based on the size of the substrate-binding pocket. Furthermore, AtSWEET13 appears to form a higher order structure presumably related to its function.

Modular architecture of eukaryotic RNase P and RNase MRP revealed by electron microscopy.

PubMed

Hipp, Katharina; Galani, Kyriaki; Batisse, Claire; Prinz, Simone; Böttcher, Bettina

2012-04-01

Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined. Using electron microscopy and image processing we show that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules. Such features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence. These are also the sites of greatest difference between RNase P and RNase MRP, highlighting the importance of the adaptation of this region to the different substrates.

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP

PubMed Central

Jaru-Ampornpan, Peera; Shen, Kuang; Lam, Vinh Q.; Ali, Mona; Doniach, Sebastian; Jia, Tony Z.; Shan, Shu-ou

2010-01-01

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and require chaperones to keep them soluble and translocation-competent. Here we show that a novel targeting factor in the chloroplast Signal Recognition Particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to AAA+-chaperones, cpSRP43 utilizes specific binding interactions with its substrate to mediate its disaggregase activity. This ‘disaggregase’ capability can allow targeting machineries to more effectively capture their protein substrates, and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example of an ATP-independent disaggregase, and demonstrates that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate. PMID:20424608

The IRS-1 signaling system.

PubMed

Myers, M G; Sun, X J; White, M F

1994-07-01

Insulin-receptor substrate 1 (IRS-1) is a principal substrate of the receptor tyrosine kinase for insulin and insulin-like growth factor 1, and a substrate for a tyrosine kinase activated by interleukin 4. IRS-1 undergoes multisite tyrosine phosphorylation and mediates downstream signals by 'docking' various proteins that contain Src homology 2 domains. IRS-1 appears to be a unique molecule; however, 4PS, a protein found mainly in hemopoietic cells, may represent another member of this family.

Structure and molecular dynamics simulation of archaeal prefoldin: the molecular mechanism for binding and recognition of nonnative substrate proteins.

PubMed

Ohtaki, Akashi; Kida, Hiroshi; Miyata, Yusuke; Ide, Naoki; Yonezawa, Akihiro; Arakawa, Takatoshi; Iizuka, Ryo; Noguchi, Keiichi; Kita, Akiko; Odaka, Masafumi; Miki, Kunio; Yohda, Masafumi

2008-02-29

Prefoldin (PFD) is a heterohexameric molecular chaperone complex in the eukaryotic cytosol and archaea with a jellyfish-like structure containing six long coiled-coil tentacles. PFDs capture protein folding intermediates or unfolded polypeptides and transfer them to group II chaperonins for facilitated folding. Although detailed studies on the mechanisms for interaction with unfolded proteins or cooperation with chaperonins of archaeal PFD have been performed, it is still unclear how PFD captures the unfolded protein. In this study, we determined the X-ray structure of Pyrococcus horikoshii OT3 PFD (PhPFD) at 3.0 A resolution and examined the molecular mechanism for binding and recognition of nonnative substrate proteins by molecular dynamics (MD) simulation and mutation analyses. PhPFD has a jellyfish-like structure with six long coiled-coil tentacles and a large central cavity. Each subunit has a hydrophobic groove at the distal region where an unfolded substrate protein is bound. During MD simulation at 330 K, each coiled coil was highly flexible, enabling it to widen its central cavity and capture various nonnative proteins. Docking MD simulation of PhPFD with unfolded insulin showed that the beta subunit is essentially involved in substrate binding and that the alpha subunit modulates the shape and width of the central cavity. Analyses of mutant PhPFDs with amino acid replacement of the hydrophobic residues of the beta subunit in the hydrophobic groove have shown that beta Ile107 has a critical role in forming the hydrophobic groove.

Laser surface alloying of coins for authenticity

NASA Astrophysics Data System (ADS)

Liu, Zhu; Watkins, Kenneth G.; Steen, William M.; Hatherley, P. G.

1997-08-01

This paper presents an exploratory investigation on verifying the feasibility of using a laser surface alloying technique to produce designs in the surface of coinage blanks. The specific aim of the work concerns the production of design features in coins that are difficult to produce by other techniques and which hence act as a barrier to forgery and features which permit automatic recognition in vending machines, particularly as a means of establishing the authenticity of the coins. Coins in many countries today are commonly manufactured from metal composites, where one substrate metal or alloy is coated with another by a process of electrodeposition or by mechanical bonding. The technique here described entails the use of a high power CO2 laser to bring about localized melting of the two layers. Visible distinction between alloyed and unalloyed regions or difference in other physical properties such as conductivity or magnetic properties can be obtained. The work also involved a fundamental study of the influence of the thermal properties of the materials on the CO2 laser alloying process. It was found that the thermal properties such as thermal conductivity of the substrate materials and the difference of the melting points between the coating layer and the substrate materials played an important role in the process. Laser control variables required for localized alloying for different substrate and coatings types were determined. The influence of both thermal properties and laser control variables on alloy type and alloy depth were investigated. Initial work on coin validation showed promising results of an automatic recognition of laser treated coins.

Biomaterial Substrate-Mediated Multicellular Spheroid Formation and Their Applications in Tissue Engineering.

PubMed

Tseng, Ting-Chen; Wong, Chui-Wei; Hsieh, Fu-Yu; Hsu, Shan-Hui

2017-12-01

Three-dimentional (3D) multicellular aggregates (spheroids), compared to the traditional 2D monolayer cultured cells, are physiologically more similar to the cells in vivo. So far there are various techniques to generate 3D spheroids. Spheroids obtained from different methods have already been applied to regenerative medicine or cancer research. Among the cell spheroids created by different methods, the substrate-derived spheroids and their forming mechanism are unique. This review focuses on the formation of biomaterial substrate-mediated multicellular spheroids and their applications in tissue engineering and tumor models. First, the authors will describe the special chitosan substrate-derived mesenchymal stem cell (MSC) spheroids and their greater regenerative capacities in various tissues. Second, the authors will describe tumor spheroids derived on chitosan and hyaluronan substrates, which serve as a simple in vitro platform to study 3D tumor models or to perform cancer drug screening. Finally, the authors will mention the self-assembly process for substrate-derived multiple cell spheroids (co-spheroids), which may recapitulate the heterotypic cell-cell interaction for co-cultured cells or crosstalk between different types of cells. These unique multicellular mono-spheroids or co-spheroids represent a category of 3D cell culture with advantages of biomimetic cell-cell interaction, better functionalities, and imaging possibilities. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Social Media: More Than Just a Communications Medium

DTIC Science & Technology

2012-03-14

video-hosting web services with the recognition that “Internet-based capabilities are integral to operations across the Department of Defense.”10...as DoD and the government as a whole, the U.S. Army’s recognition of social media’s unique relationship to time and speed is a step forward toward...populated size of social media entities, Alexa , the leader in free global web analytics, provides an updated list of the top 500 websites on the Internet

Label-free SERS detection of Salmonella Typhimurium on DNA aptamer modified AgNR substrates

USDA-ARS?s Scientific Manuscript database

Salmonella Typhimurium is an important foodborne pathogen which causes gastroenteritis in both humans and animals. Currently available rapid methods have relied on antibodies to offer specific recognition of the pathogen from the background. As a substitute of antibodies, nucleic acid aptamers offer...

Object Recognition and Random Image Structure Evolution

ERIC Educational Resources Information Center

Sadr, Jvid; Sinha, Pawan

2004-01-01

We present a technique called Random Image Structure Evolution (RISE) for use in experimental investigations of high-level visual perception. Potential applications of RISE include the quantitative measurement of perceptual hysteresis and priming, the study of the neural substrates of object perception, and the assessment and detection of subtle…

Decoding the patterns of ubiquitin recognition by ubiquitin-associated domains from free energy simulations.

PubMed

Bouvier, Benjamin

2014-01-07

Ubiquitin is a highly conserved, highly represented protein acting as a regulating signal in numerous cellular processes. It leverages a single hydrophobic binding patch to recognize and bind a large variety of protein domains with remarkable specificity, but can also self-assemble into chains of poly-diubiquitin units in which these interfaces are sequestered, profoundly altering the individual monomers' recognition characteristics. Despite numerous studies, the origins of this varied specificity and the competition between substrates for the binding of the ubiquitin interface patch remain under heated debate. This study uses enhanced sampling all-atom molecular dynamics to simulate the unbinding of complexes of mono- or K48-linked diubiquitin bound to several ubiquitin-associated domains, providing insights into the mechanism and free energetics of ubiquitin recognition and binding. The implications for the subtle tradeoff between the stability of the polyubiquitin signal and its easy recognition by target protein assemblies are discussed, as is the enhanced affinity of the latter for long polyubiquitin chains compared to isolated mono- or diubiquitin.

Molecular recognition on a cavitand-functionalized silicon surface.

PubMed

Biavardi, Elisa; Favazza, Maria; Motta, Alessandro; Fragalà, Ignazio L; Massera, Chiara; Prodi, Luca; Montalti, Marco; Melegari, Monica; Condorelli, Guglielmo G; Dalcanale, Enrico

2009-06-03

A Si(100) surface featuring molecular recognition properties was obtained by covalent functionalization with a tetraphosphonate cavitand (Tiiii), able to complex positively charged species. Tiiii cavitand was grafted onto the Si by photochemical hydrosilylation together with 1-octene as a spatial spectator. The recognition properties of the Si-Tiiii surface were demonstrated through two independent analytical techniques, namely XPS and fluorescence spectroscopy, during the course of reversible complexation-guest exchange-decomplexation cycles with specifically designed ammonium and pyridinium salts. Control experiments employing a Si(100) surface functionalized with a structurally similar, but complexation inactive, tetrathiophosphonate cavitand (TSiiii) demonstrated no recognition events. This provides evidence for the complexation properties of the Si-Tiiii surface, ruling out the possibility of nonspecific interactions between the substrate and the guests. The residual Si-O(-) terminations on the surface replace the guests' original counterions, thus stabilizing the complex ion pairs. These results represent a further step toward the control of self-assembly of complex supramolecular architectures on surfaces.

"Off-On"switching electrochemiluminescence biosensor for mercury(II) detection based on molecular recognition technology.

PubMed

Cheng, Lin; Wei, BingGuo; He, Ling Ling; Mao, Ling; Zhang, Jie; Ceng, JinXiang; Kong, DeRong; Chen, ChaDan; Cui, HanFeng; Hong, Nian; Fan, Hao

2017-02-01

A novel "off-On" electrogenerated chemiluminescence (ECL) biosensor has been developed for the detection of mercury(II) based on molecular recognition technology. The ECL mercury(II) biosensor comprises two main parts: an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Ruthenium(II) tris-(bipyridine)(Ru(bpy) 3 2+ )/Cyclodextrins-Au nanoparticles(CD-AuNps)/Nafion on the surface of glass carbon electrode (GCE), and the ECL intensity switch is the single hairpin DNA probe designed according to the "molecular recognition" strategy which was functionalized with ferrocene tag at one end and attached to Cyclodextrins (CD) on modified GCE through supramolecular noncovalent interaction. We demonstrated that, in the absence of Hg(II) ion, the probe keeps single hairpin structure and resulted in a quenching of ECL of Ru(bpy) 3 2+ . Whereas, in the presence of Hg(II) ion, the probe prefers to form the T-Hg(II)-T complex and lead to an obvious recovery of ECL of Ru(bpy) 3 2+ , which provided a sensing platform for the detection of Hg(II) ion. Using this sensing platform, a simple, rapid and selective "off-On" ECL biosensor for the detection of mercury(II) with a detection limit of 0.1 nM has been developed. Copyright © 2016. Published by Elsevier Inc.

Minimal sulfated carbohydrates for recognition by L-selectin and the MECA-79 antibody.

PubMed

Bruehl, R E; Bertozzi, C R; Rosen, S D

2000-10-20

Sulfated forms of sialyl-Le(X) containing Gal-6-SO(4) or GlcNAc-6-SO(4) have been implicated as potential recognition determinants on high endothelial venule ligands for L-selectin. The optimal configuration of sulfate esters on the N-acetyllactosamine (Galbeta1-->4GlcNAc) core of sulfosialyl-Le(X), however, remains unsettled. Using a panel of sulfated lactose (Galbeta1-->4Glc) neoglycolipids as substrates in direct binding assays, we found that 6',6-disulfolactose was the preferred structure for L-selectin, although significant binding to 6'- and 6-sulfolactose was observed as well. Binding was EDTA-sensitive and blocked by L-selectin-specific monoclonal antibodies. Surprisingly, 6', 6-disulfolactose was poorly recognized by MECA-79, a carbohydrate- and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Instead, MECA-79 bound preferentially to 6-sulfolactose. The difference in preferred substrates between L-selectin and MECA-79 may explain the variable activity of MECA-79 as an inhibitor of lymphocyte adhesion to high endothelial venules in lymphoid organs. Our results suggest that both Gal-6-SO(4) and GlcNAc-6-SO(4) may contribute to L-selectin recognition, either as components of sulfosialyl-Le(X) capping groups or in internal structures. By contrast, only GlcNAc-6-SO(4) appears to contribute to MECA-79 binding.

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohman, Jeremy; Ma, Ming; Osipiuk, Jerzy

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representationmore » of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.« less

Eu3+-doped Gd2O3 nanoparticles as reporters for optical detection and visualization of antibodies patterned by microcontact printing.

PubMed

Nichkova, Mikaela; Dosev, Dosi; Perron, Richard; Gee, Shirley J; Hammock, Bruce D; Kennedy, Ian M

2006-02-01

Lanthanide oxide nanoparticles are promising luminescent probes in bioanalysis, because of their unique spectral properties, photostability, and low-cost synthesis. We report for the first time the application of europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles to the optical imaging of antibody micropatterns. The nanoparticles were synthesized by spray pyrolysis and coated with antibody (IgG) molecules by physical adsorption. Our experiments showed that the Eu:Gd2O3 is a good biocompatible solid support for antibody immobilization. The antibodies (anti-rabbit IgG) immobilized on the nanoparticles had excellent biological activity in the specific recognition reaction with rabbit IgG patterned in line strips (10 micromx10 microm) on a glass substrate by use of a micro-contact printing technique. The specific immunoreaction was confirmed by two independent microscopic techniques-fluorescence and scanning electron microscopy (SEM). Both microscopic images revealed that the nanoparticles were organized into designated structures as defined by the microcontact printing process with negligible non-specific binding. The nanoparticles can be used as fluorescent markers in a variety of immunosensing applications in a microscale format.

Cytosolic Hsp70 and co-chaperones constitute a novel system for tRNA import into the nucleus

PubMed Central

Takano, Akira; Kajita, Takuya; Mochizuki, Makoto; Endo, Toshiya; Yoshihisa, Tohru

2015-01-01

tRNAs are unique among various RNAs in that they shuttle between the nucleus and the cytoplasm, and their localization is regulated by nutrient conditions. Although nuclear export of tRNAs has been well documented, the import machinery is poorly understood. Here, we identified Ssa2p, a major cytoplasmic Hsp70 in Saccharomyces cerevisiae, as a tRNA-binding protein whose deletion compromises nuclear accumulation of tRNAs upon nutrient starvation. Ssa2p recognizes several structural features of tRNAs through its nucleotide-binding domain, but prefers loosely-folded tRNAs, suggesting that Ssa2p has a chaperone-like activity for RNAs. Ssa2p also binds Nup116, one of the yeast nucleoporins. Sis1p and Ydj1p, cytoplasmic co-chaperones for Ssa proteins, were also found to contribute to the tRNA import. These results unveil a novel function of the Ssa2p system as a tRNA carrier for nuclear import by a novel mode of substrate recognition. Such Ssa2p-mediated tRNA import likely contributes to quality control of cytosolic tRNAs. DOI: http://dx.doi.org/10.7554/eLife.04659.001 PMID:25853343

Chymotryptic specificity determinants in the 1.0 Å structure of the zinc-inhibited human tissue kallikrein 7

PubMed Central

Debela, Mekdes; Hess, Petra; Magdolen, Viktor; Schechter, Norman M.; Steiner, Thomas; Huber, Robert; Bode, Wolfram; Goettig, Peter

2007-01-01

hK7 or human stratum corneum chymotryptic enzyme belongs to the human tissue kallikrein (hKs) serine proteinase family and is strongly expressed in the upper layers of the epidermis. It participates in skin desquamation but is also implicated in diverse skin diseases and is a potential biomarker of ovarian cancer. We have solved x-ray structures of recombinant active hK7 at medium and atomic resolution in the presence of the inhibitors succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone and Ala-Ala-Phe-chloromethyl ketone. The most distinguishing features of hK7 are the short 70–80 loop and the unique S1 pocket, which prefers P1 Tyr residues, as shown by kinetic data. Similar to several other kallikreins, the enzyme activity is inhibited by Zn2+ and Cu2+ at low micromolar concentrations. Biochemical analyses of the mutants H99A and H41F confirm that only the metal-binding site at His99 close to the catalytic triad accounts for the noncompetitive Zn2+ inhibition type. Additionally, hK7 exhibits large positively charged surface patches, representing putative exosites for prime side substrate recognition. PMID:17909180

Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana

PubMed Central

Rustgi, Sachin; Boex-Fontvieille, Edouard; Reinbothe, Christiane; von Wettstein, Diter; Reinbothe, Steffen

2017-01-01

Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21’s activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening. PMID:28179567

Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana.

PubMed

Rustgi, Sachin; Boex-Fontvieille, Edouard; Reinbothe, Christiane; von Wettstein, Diter; Reinbothe, Steffen

2017-02-28

Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21's activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening.

Effects of Combinations of Substrates on Maximum Growth Rates of Several Rumen Bacteria

PubMed Central

Russell, James B.; Delfino, Frank J.; Baldwin, R. L.

1979-01-01

Five rumen bacteria, Selenomonas ruminantium, Bacteroides ruminicola, Megasphaera elsdenii, Butyrivibrio fibrisolvens, and Streptococcus bovis were grown in media containing nonlimiting concentrations of glucose, sucrose, maltose, cellobiose, xylose and/or lactate. Each bacterium was grown with every substrate that it could ferment in every possible two-way combination. Only once did a combination of substrates result in a higher maximum growth rate than that observed with either substrate alone. Such stimulations of growth rate would be expected if specific factors unique to individual substrates (transport proteins and/or enzymes) were limiting. Since such synergisms were rare, it was concluded that more general factors limit maximum growth rates in these five bacteria. PMID:16345360

Studies of lysine cyclodeaminase from Streptomyces pristinaespiralis: Insights into the complex transition NAD+ state.

PubMed

Ying, Hanxiao; Wang, Jing; Shi, Ting; Zhao, Yilei; Wang, Xin; Ouyang, Pingkai; Chen, Kequan

2018-01-01

Lysine cyclodeaminase (LCD) catalyzes the piperidine ring formation in macrolide-pipecolate natural products metabolic pathways from a lysine substrate through a combination of cyclization and deamination. This enzyme belongs to a unique enzyme class, which uses NAD + as the catalytic prosthetic group instead of as the co-substrate. To understand the molecular details of NAD + functions in lysine cyclodeaminase, we have determined four ternary crystal structure complexes of LCD-NAD + with pipecolic acid (LCD-PA), lysine (LCD-LYS), and an intermediate (LCD-INT) as ligands at 2.26-, 2.00-, 2.17- and 1.80 Å resolutions, respectively. By combining computational studies, a NAD + -mediated "gate keeper" function involving NAD + /NADH and Arg49 that control the binding and entry of the ligand lysine was revealed, confirming the critical roles of NAD + in the substrate access process. Further, in the gate opening form, a substrate delivery tunnel between ε-carboxyl moiety of Glu264 and the α-carboxyl moiety of Asp236 was observed through a comparison of four structure complexes. The LCD structure details including NAD + -mediated "gate keeper" and substrate tunnel may assist in the exploration the NAD + function in this unique enzyme class, and in regulation of macrolide-pipecolate natural product synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Direct Role for Proliferating Cell Nuclear Antigen in Substrate Recognition by the E3 Ubiquitin Ligase CRL4Cdt2*

PubMed Central

Havens, Courtney G.; Shobnam, Nadia; Guarino, Estrella; Centore, Richard C.; Zou, Lee; Kearsey, Stephen E.; Walter, Johannes C.

2012-01-01

The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4Cdt2) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4Cdt2 substrates contain a “PIP degron,” which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4Cdt2 for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4Cdt2 substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4Cdt2 recruitment to chromatin. Our data show that the interaction of CRL4Cdt2 with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions. PMID:22303007

Temperature Dependent Performance of Coplanar Waveguide (CPW) on Substrates of Various Materials

NASA Technical Reports Server (NTRS)

Taub, Susan R.; Young, Paul

1994-01-01

The attenuation (a) and effective dielectric constant (E(sub eff)) of Coplanar Waveguide (CPW) transmission lines on high-resistivity silicon and diamond substrates as a function of both temperature and frequency are presented. The technique used to obtain the values for a and E(sub eff) involves the use of a unique cryogenic probe station designed and built by NASA. Attenuation of gold CPW lines on diamond substrates is compared with that of superconducting CPW lines.

Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes*

PubMed Central

Nam, Young-Woo; Nihira, Takanori; Arakawa, Takatoshi; Saito, Yuka; Kitaoka, Motomitsu; Nakai, Hiroyuki; Fushinobu, Shinya

2015-01-01

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes. PMID:26041776

Lead discovery and chemical biology approaches targeting the ubiquitin proteasome system.

PubMed

Akinjiyan, Favour A; Carbonneau, Seth; Ross, Nathan T

2017-10-15

Protein degradation is critical for proteostasis, and the addition of polyubiquitin chains to a substrate is necessary for its recognition by the 26S proteasome. Therapeutic intervention in the ubiquitin proteasome system has implications ranging from cancer to neurodegeneration. Novel screening methods and chemical biology tools for targeting E1-activating, E2-conjugating and deubiquitinating enzymes will be discussed in this review. Approaches for targeting E3 ligase-substrate interactions as well as the proteasome will also be covered, with a focus on recently described approaches. Copyright © 2017. Published by Elsevier Ltd.

Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

PubMed Central

Xu, Yingqi; Plechanovová, Anna; Simpson, Peter; Marchant, Jan; Leidecker, Orsolya; Kraatz, Sebastian; Hay, Ronald T.; Matthews, Steve J.

2014-01-01

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer. PMID:24969970

Miscoding-induced stalling of substrate translocation on the bacterial ribosome.

PubMed

Alejo, Jose L; Blanchard, Scott C

2017-10-10

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G-catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.

Miscoding-induced stalling of substrate translocation on the bacterial ribosome

PubMed Central

Alejo, Jose L.; Blanchard, Scott C.

2017-01-01

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors. PMID:28973849

Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base-Modified Nucleosides.

PubMed

Fateev, Ilja V; Kharitonova, Maria I; Antonov, Konstantin V; Konstantinova, Irina D; Stepanenko, Vasily N; Esipov, Roman S; Seela, Frank; Temburnikar, Kartik W; Seley-Radtke, Katherine L; Stepchenko, Vladimir A; Sokolov, Yuri A; Miroshnikov, Anatoly I; Mikhailopulo, Igor A

2015-09-14

A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild-type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8-aza-7-deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α-D-pentofuranose-1-phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2-deoxy-α-D-ribofuranose-1-phosphate in the trans-2-deoxyribosylation reaction. 5-Aza-7-deazaguanine manifested excellent substrate activity for both enzymes, 8-amino-7-thiaguanine and 2-aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2-amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1- and unusual N2-glycosides, respectively. 9-Deaza-5-iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9-deazaxanthine and its 2'-deoxyriboside are weak inhibitors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Robust Radio Broadcast Monitoring Using a Multi-Band Spectral Entropy Signature

NASA Astrophysics Data System (ADS)

Camarena-Ibarrola, Antonio; Chávez, Edgar; Tellez, Eric Sadit

Monitoring media broadcast content has deserved a lot of attention lately from both academy and industry due to the technical challenge involved and its economic importance (e.g. in advertising). The problem pose a unique challenge from the pattern recognition point of view because a very high recognition rate is needed under non ideal conditions. The problem consist in comparing a small audio sequence (the commercial ad) with a large audio stream (the broadcast) searching for matches.

Carbon dioxide-expanded liquid substrate phase: an effective medium for selective hydrogenation of cinnamaldehyde to cinnamyl alcohol.

PubMed

Zhao, Fengyu; Fujita, Shin-ichio; Sun, Jianmin; Ikushima, Yutaka; Arai, Masahiko

2004-10-21

It has been shown that CO(2)-expanded cinnamaldehyde liquid phase is a unique and effective medium for cinnamaldehyde hydrogenation to cinnamyl alcohol, due to interactions between the C[double bond, length as m-dash]O group of the substrate and CO(2) molecules and increased solubility of H(2).

Surface wave chemical detector using optical radiation

DOEpatents

Thundat, Thomas G.; Warmack, Robert J.

2007-07-17

A surface wave chemical detector comprising at least one surface wave substrate, each of said substrates having a surface wave and at least one measurable surface wave parameter; means for exposing said surface wave substrate to an unknown sample of at least one chemical to be analyzed, said substrate adsorbing said at least one chemical to be sensed if present in said sample; a source of radiation for radiating said surface wave substrate with different wavelengths of said radiation, said surface wave parameter being changed by said adsorbing; and means for recording signals representative of said surface wave parameter of each of said surface wave substrates responsive to said radiation of said different wavelengths, measurable changes of said parameter due to adsorbing said chemical defining a unique signature of a detected chemical.

L2 Word Recognition: Influence of L1 Orthography on Multi-syllabic Word Recognition.

PubMed

Hamada, Megumi

2017-10-01

L2 reading research suggests that L1 orthographic experience influences L2 word recognition. Nevertheless, the findings on multi-syllabic words in English are still limited despite the fact that a vast majority of words are multi-syllabic. The study investigated whether L1 orthography influences the recognition of multi-syllabic words, focusing on the position of an embedded word. The participants were Arabic ESL learners, Chinese ESL learners, and native speakers of English. The task was a word search task, in which the participants identified a target word embedded in a pseudoword at the initial, middle, or final position. The search accuracy and speed indicated that all groups showed a strong preference for the initial position. The accuracy data further indicated group differences. The Arabic group showed higher accuracy in the final than middle, while the Chinese group showed the opposite and the native speakers showed no difference between the two positions. The findings suggest that L2 multi-syllabic word recognition involves unique processes.

Unique Ganglioside Recognition Strategies for Clostridial Neurotoxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benson, Marc A.; Fu, Zhuji; Kim, Jung-Ja P.

2012-03-15

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin are the causative agents of the paralytic diseases botulism and tetanus, respectively. The potency of the clostridial neurotoxins (CNTs) relies primarily on their highly specific binding to nerve terminals and cleavage of SNARE proteins. Although individual CNTs utilize distinct proteins for entry, they share common ganglioside co-receptors. Here, we report the crystal structure of the BoNT/F receptor-binding domain in complex with the sugar moiety of ganglioside GD1a. GD1a binds in a shallow groove formed by the conserved peptide motif E ... H ... SXWY ... G, with additional stabilizing interactions provided by two argininemore » residues. Comparative analysis of BoNT/F with other CNTs revealed several differences in the interactions of each toxin with ganglioside. Notably, exchange of BoNT/F His-1241 with the corresponding lysine residue of BoNT/E resulted in increased affinity for GD1a and conferred the ability to bind ganglioside GM1a. Conversely, BoNT/E was not able to bind GM1a, demonstrating a discrete mechanism of ganglioside recognition. These findings provide a structural basis for ganglioside binding among the CNTs and show that individual toxins utilize unique ganglioside recognition strategies.« less

Improved microautoradiographic method to determine individual microorganisms active in substrate uptake in natural waters.

PubMed

Tabor, P S; Neihof, R A

1982-10-01

We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method.

Improved Microautoradiographic Method to Determine Individual Microorganisms Active in Substrate Uptake in Natural Waters

PubMed Central

Tabor, Paul S.; Neihof, Rex A.

1982-01-01

We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with 3H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (3H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method. Images PMID:16346120

Identification and Targeting of Tyrosine Kinase Activity in Prostate Cancer Initiation, Progression, and Metastasis

DTIC Science & Technology

2013-12-01

University) "Effectors of the DNA damage and radiotherapy response in cancer" 9:20 pm - 9:30 pm Discussion TUESDAY 7:30 am - 8:30 am Breakfast 9:00...M. Morris , Hideki Onagi, Timothy M. Altamore, Allan B. Gamble, Christopher J. Easton Prohormone-substrate peptide sequence recognition by

Soil fauna and plant litter decomposition in tropical and subalpine forests

Treesearch

G. Gonzalez; T.R. Seastedt

2001-01-01

The decomposition of plant residues is influenced by their chemical composition, the physical-chemical environment, and the decomposer organisms. Most studies interested in latitudinal gradients of decomposition have focused on substrate quality and climate effects on decomposition, and have excluded explicit recognition of the soil organisms involved in the process....

Brain catechol-o-methyltransferase (COMT) inhibition by tolcapone counteracts recognition memory deficits in normal and chronic phencyclidine-treated rats and in COMT-Val transgenic mice

PubMed Central

Detrait, E.R.; Carr, G.V.; Ferraille, S.; Weinberger, D.R.; Lamberty, Y.

2015-01-01

The critical involvement of dopamine in cognitive processes has been well established, suggesting therapies targeting dopamine metabolism may alleviate cognitive dysfunction. COMT is a catecholamine-degrading enzyme, the substrates of which include dopamine, epinephrine, and norepinephrine. The present work illustrates the potential therapeutic efficacy of COMT inhibition for alleviating cognitive impairment. A brain penetrant COMT inhibitor, tolcapone, was tested in normal and phencyclidine (PCP)-treated rats and COMT–Val transgenic mice. In a Novel Object Recognition (NOR) procedure, tolcapone counteracted a 24h-dependent forgetting of a familiar object and counteracted PCP-induced recognition deficits in the rats at doses ranging from 7.5 to 30 mg/kg. In contrast, entacapone, a COMT inhibitor which does not readily cross the blood-brain barrier failed to show efficacy at doses up to 30mg/kg. Tolcapone at a dose of 30 mg/kg also improved NOR performance in the transgenic mice, which showed clear recognition deficits. Complementing earlier studies, our results indicate that central inhibition of COMT positively impacts recognition memory processes and might constitute an appealing treatment for cognitive dysfunction related to neuropsychiatric disorders. PMID:26919286

Supramolecular control of transition metal complexes in water by a hydrophobic cavity: a bio-inspired strategy.

PubMed

Bistri, Olivia; Reinaud, Olivia

2015-03-14

Supramolecular chemistry in water is a very challenging research area. In biology, water is the universal solvent where transition metal ions play major roles in molecular recognition and catalysis. In enzymes, it participates in substrate binding and/or activation in the heart of a pocket defined by the folded protein. The association of a hydrophobic cavity with a transition metal ion is thus a very appealing strategy for controlling the metal ion properties in the very competitive water solvent. Various systems based on intrinsically water-soluble macrocyclic structures such as cyclodextrins, cucurbituryls, and metallo-cages have been reported. Others use calixarenes and resorcinarenes functionalized with hydrophilic substituents. One approach for connecting a metal complex to these cavities is to graft a ligand for metal ion binding at their edge. Early work with cyclodextrins has shown Michaelis-Menten like catalysis displaying enhanced kinetics and substrate-selectivity. Remarkable examples of regio- and stereo-selective transformation of substrates have been reported as well. Dynamic two-phase systems for transition metal catalysis have also been developed. They rely on either water-transfer of the metal complex through ligand embedment or synergistic coordination of a metal ion and substrate hosting. Another strategy consists in using metallo-cages, which provide a well-defined hydrophobic space, to stabilize metal complexes in water. When the cages can host simultaneously a substrate and a reactive metal complex, size- and regio-selective catalysis was obtained. Finally, construction of a polydentate coordination site closely interlocked with a calixarene or resorcinarene macrocycle has been shown to be a very fruitful strategy for obtaining metal complexes with remarkable hosting properties. For each of these systems, the synergism resulting from the biomimetic association of a hydrophobic cavity and a metal ion is discussed within the objective of developing new tools for either selective molecular recognition (with analytical perspectives) or performant catalysis, in water.

Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

PubMed Central

Singh Gautam, Amit Kumar; Balakrishnan, Satish; Venkatraman, Prasanna

2012-01-01

Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradation PMID:22506054

Band gap modulation of graphene by metal substrate: A first principles study

NASA Astrophysics Data System (ADS)

Sahoo, Mihir Ranjan; Sahu, Sivabrata; Kushwaha, Anoop Kumar; Nayak, S. K.

2018-04-01

Due to high in-plane charge carrier mobility with high electron velocity and long spin diffusion length, graphene guarantees as a completely unique material for devices with various applications. Unaffected 2pz orbitals of carbon atoms in graphene can be highly influenced by substrates and leads to tuning in electronic properties. We report here a density functional calculation of graphene monolayer based on metallic substrate like nickel surfaces. Band-gap of graphene near K points opens due to interactions between 2pz and d-orbitals of nickel atoms and the gap modulation can be done with the increasing number of layers of substrates.

Caspases and Kinases in a Death Grip

PubMed Central

Kurokawa, Manabu; Kornbluth, Sally

2011-01-01

The complex process of apoptosis is orchestrated by caspases, a family of cysteine proteases with unique substrate specificities. Accumulating evidence suggests that cell death pathways are finely tuned by multiple signaling events, including direct phosphorylation of caspases, whereas kinases are often substrates of active caspases. Importantly, caspase-mediated cleavage of kinases can terminate prosurvival signaling or generate proapoptotic peptide fragments that help to execute the death program and facilitate packaging of the dying cells. Here, we review caspases as kinase substrates and kinases as caspase substrates and discuss how the balance between cell survival and cell death can be shifted through crosstalk between these two enzyme families. PMID:19737514

γ-secretase composed of PS1/Pen2/Aph1a can cleave Notch and APP in the absence of Nicastrin

PubMed Central

Zhao, Guojun; Liu, Zhenyi; Ilagan, Ma. Xenia G.; Kopan, Raphael

2010-01-01

γ-secretase is a multiprotein intramembrane-cleaving protease with a growing list of protein substrates including the Notch receptors and the amyloid precursor protein. The four components of γ-secretase complex - presenilin (PS), nicastrin (NCT), Pen2, and Aph1 - are all thought to be essential for activity. The catalytic domain resides within PS proteins; NCT has been suggested to be critical for substrate recognition; the contributions of Pen2 and Aph1 remain unclear. The role of NCT has been challenged recently by the observation that a critical residue (E332) in NCT, thought to be essential for γ-secretase activity, is instead involved in complex maturation. Here we report that NCT is dispensable for γ-secretase activity. NCT-independent γ-secretase activity can be detected in two independent NCT-deficient MEF lines, and blocked by the γ-secretase inhibitors DAPT and L-685,458. This catalytic activity requires prior ectodomain shedding of the substrate, and can cleave ligand-activated endogenous Notch receptors, indicating presence at the plasma membrane. siRNA knockdown experiments demonstrated that NCT-independent γ-secretase activity requires the presence of PS1, Pen2 and Aph1a but can tolerate knockdown of PS2 or Aph1b. We conclude that a PS1/Pen2/Aph1a trimeric complex is an active enzyme, displaying similar biochemical properties to those of γ-secretase and roughly 50% of its activity when normalized to PS1 NTF levels. This PS1/Pen2/Aph1a complex, however, is highly unstable. Thus, NCT acts to stabilize γ-secretase, but is not required for substrate recognition. PMID:20130175

Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast

PubMed Central

Maurer, Matthew J.; Spear, Eric D.; Yu, Allen T.; Lee, Evan J.; Shahzad, Saba; Michaelis, Susan

2016-01-01

Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic “degron library” in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3. About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation.

PubMed

Ninio, Shira; Zuckman-Cholon, Deborah M; Cambronne, Eric D; Roy, Craig R

2005-02-01

The intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella, the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two-hybrid screen. It was determined that the IcmS-IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS-IcmW complex being involved in the recognition and Dot/Icm-dependent translocation of substrate proteins during Legionella infection of host cells.

Substrate-induced ubiquitylation and endocytosis of yeast amino acid permeases.

PubMed

Ghaddar, Kassem; Merhi, Ahmad; Saliba, Elie; Krammer, Eva-Maria; Prévost, Martine; André, Bruno

2014-12-01

Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates

PubMed Central

Gwon, Gwang Hyeon; Jo, Aera; Baek, Kyuwon; Jin, Kyeong Sik; Fu, Yaoyao; Lee, Jong-Bong; Kim, YoungChang; Cho, Yunje

2014-01-01

The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3′ flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a “5′ end binding pocket” that hosts the 5′ nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3′ flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5′ flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3′ flap DNA substrates with 5′ nicked ends. PMID:24733841

Deciphering kinase-substrate relationships by analysis of domain-specific phosphorylation network.

PubMed

Damle, Nikhil Prakash; Mohanty, Debasisa

2014-06-15

In silico prediction of site-specific kinase-substrate relationships (ssKSRs) is crucial for deciphering phosphorylation networks by linking kinomes to phosphoproteomes. However, currently available predictors for ssKSRs give rise to a large number of false-positive results because they use only a short sequence stretch around phosphosite as determinants of kinase specificity and do not consider the biological context of kinase-substrate recognition. Based on the analysis of domain-specific kinase-substrate relationships, we have constructed a domain-level phosphorylation network that implicitly incorporates various contextual factors. It reveals preferential phosphorylation of specific domains by certain kinases. These novel correlations have been implemented in PhosNetConstruct, an automated program for predicting target kinases for a substrate protein. PhosNetConstruct distinguishes cognate kinase-substrate pairs from a large number of non-cognate combinations. Benchmarking on independent datasets using various statistical measures demonstrates the superior performance of PhosNetConstruct over ssKSR-based predictors. PhosNetConstruct is freely available at http://www.nii.ac.in/phosnetconstruct.html. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif

PubMed Central

Song, Ruiwen; Li, Jing; Zhang, Jin; Wang, Lu; Tong, Li; Wang, Ping; Yang, Huan; Wei, Qun; Cai, Huaibin; Luo, Jing

2018-01-01

Calcineurin (CN) is involved in many physiological processes and interacts with multiple substrates. Most of the substrates contain similar motifs recognized by CN. Recent studies revealed a new CN substrate, transcription factor EB (TFEB), which is involved in autophagy. We showed that a 15-mer QSYLENPTSYHLQQS peptide from TFEB (TFEB-YLENP) bound to CN. When the TFEB-YLENP peptide was changed to YLAVP, its affinity for CN increased and it had stronger CN inhibitory activity. Molecular dynamics simulations revealed that the TFEB-YLENP peptide has the same docking sites in CN as the 15-mer DQYLAVPQHPYQWAK motif of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1-YLAVP). Moreover expression of the NFATc1-YLAVP peptide suppressed the TFEB activation in starved Hela cells. Our studies first identified a CN binding site in TFEB and compared the inhibitory capability of various peptides derived from CN substrates. The data uncovered a diversity in recognition sequences that underlies the CN signaling within the cell. Studies of CN-substrate interactions should lay the groundwork for developing selective CN peptide inhibitors that target CN-substrate interaction in vitro experiments. PMID:28890387

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

PubMed Central

Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

2015-01-01

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2013-01-01

Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available. PMID:24244149

Artificially intelligent recognition of Arabic speaker using voice print-based local features

NASA Astrophysics Data System (ADS)

Mahmood, Awais; Alsulaiman, Mansour; Muhammad, Ghulam; Akram, Sheeraz

2016-11-01

Local features for any pattern recognition system are based on the information extracted locally. In this paper, a local feature extraction technique was developed. This feature was extracted in the time-frequency plain by taking the moving average on the diagonal directions of the time-frequency plane. This feature captured the time-frequency events producing a unique pattern for each speaker that can be viewed as a voice print of the speaker. Hence, we referred to this technique as voice print-based local feature. The proposed feature was compared to other features including mel-frequency cepstral coefficient (MFCC) for speaker recognition using two different databases. One of the databases used in the comparison is a subset of an LDC database that consisted of two short sentences uttered by 182 speakers. The proposed feature attained 98.35% recognition rate compared to 96.7% for MFCC using the LDC subset.

Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

PubMed

Bezerra, Gustavo Arruda; Ohara-Nemoto, Yuko; Cornaciu, Irina; Fedosyuk, Sofiya; Hoffmann, Guillaume; Round, Adam; Márquez, José A; Nemoto, Takayuki K; Djinović-Carugo, Kristina

2017-06-06

Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease.

PubMed

Rampello, Anthony J; Glynn, Steven E

2017-03-24

The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cleavage Entropy as Quantitative Measure of Protease Specificity

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

2013-01-01

A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture.

PubMed

Lacroix-Labonté, Julie; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

2012-03-01

Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure-function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing-loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (k(cat)/K(M)) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

Tailored nanoporous coatings fabricated on conformable polymer substrates.

PubMed

Poxson, David J; Mont, Frank W; Cho, Jaehee; Schubert, E Fred; Siegel, Richard W

2012-11-01

Nanoporous coatings have become the subject of intense investigation, in part because they have been shown to have unique and tailorable physical properties that can depart greatly from their dense or macroscopic counterparts. Nanoporous coatings are frequently fabricated utilizing oblique-angle or glancing-angle physical vapor-phase deposition techniques. However, a significant limitation for such coatings exists; they are almost always deposited on smooth and rigid planar substrates, such as silicon and glass. This limitation greatly constrains the applicability, tailorability, functionality and even the economic viability, of such nanoporous coatings. Here, we report our findings on nanoporous/polymer composite systems (NPCS) fabricated by utilizing oblique-angle electron-beam methodology. These unique composite systems exhibit several favorable characteristics, namely, (i) fine-tuned control over coating nanoporosity and thickness, (ii) excellent adhesion between the nanoporous coating and polymer substrate, (iii) the ability to withstand significant and repeated bending, and (iv) the ability to be molded conformably on two and three-dimensional surfaces while closely retaining the composite system's designed nanoporous film structure and, hence, properties.

Structural basis for catalysis at the membrane-water interface.

PubMed

Dufrisne, Meagan Belcher; Petrou, Vasileios I; Clarke, Oliver B; Mancia, Filippo

2017-11-01

The membrane-water interface forms a uniquely heterogeneous and geometrically constrained environment for enzymatic catalysis. Integral membrane enzymes sample three environments - the uniformly hydrophobic interior of the membrane, the aqueous extramembrane region, and the fuzzy, amphipathic interfacial region formed by the tightly packed headgroups of the components of the lipid bilayer. Depending on the nature of the substrates and the location of the site of chemical modification, catalysis may occur in each of these environments. The availability of structural information for alpha-helical enzyme families from each of these classes, as well as several beta-barrel enzymes from the bacterial outer membrane, has allowed us to review here the different ways in which each enzyme fold has adapted to the nature of the substrates, products, and the unique environment of the membrane. Our focus here is on enzymes that process lipidic substrates. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Ji-Hye; Lee, Heeseob; Kim, Young-Wan

2009-01-09

A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both {alpha}-(1,6)- and {alpha}-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with {sup 14}C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such asmore » maltononaose and maltooctaosyl-{alpha}-(1,6)-{beta}-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.« less

Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes

NASA Astrophysics Data System (ADS)

Carlson, Jacob C.; Li, Shengying; Gunatilleke, Shamila S.; Anzai, Yojiro; Burr, Douglas A.; Podust, Larissa M.; Sherman, David H.

2011-08-01

Elucidation of natural product biosynthetic pathways provides important insights into the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here, we show full reconstitution, in vitro, of an unusual multi-step oxidative cascade for post-assembly-line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and a flavin adenine dinucleotide-dependent oxidase (TamL), which act co-dependently through the repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a previously unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C10 oxidation of TirE.

Programmable RNA recognition and cleavage by CRISPR/Cas9.

PubMed

O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

2014-12-11

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

Simple and rapid mercury ion selective electrode based on 1-undecanethiol assembled Au substrate and its recognition mechanism.

PubMed

Li, Xian-Qing; Liang, Hai-Qing; Cao, Zhong; Xiao, Qing; Xiao, Zhong-Liang; Song, Liu-Bin; Chen, Dan; Wang, Fu-Liang

2017-03-01

A simple and rapid mercury ion selective electrode based on 1-undecanethiol (1-UDT) assembled Au substrate (Au/1-UDT) has been well constructed. 1-UDT was for the purpose of generating self-assembled monolayer on gold surface to recognize Hg 2+ in aqueous solution, which had a working concentration range of 1.0×10 -8 -1.0×10 -4 molL -1 , with a Nernst response slope of 28.83±0.4mV/-pC, a detection limit of 4.5×10 -9 molL -1 , and a good selectivity over the other tested cations. Also, the Au/1-UDT possessed good reproducibility, stability, and short response time. The recovery obtained for the determination of mercury ion in practical tremella samples was in the range of 99.8-103.4%. Combined electrochemical analysis and X-ray photoelectron spectroscopy (XPS) with quantum chemical computation, the probable recognition mechanism of the electrode for selective recognition of Hg 2+ has been investigated. The covalent bond formed between mercury and sulfur is stronger than the one between gold and sulfur and thus prevents the adsorption of 1-UDT molecules on the gold surface. The quantum chemical computation with density functional theory further demonstrates that the strong interaction between the mercury atom and the sulfur atom on the gold surface leads to the gold sulfur bond ruptured and the gold mercury metallophilic interaction. Copyright © 2016 Elsevier B.V. All rights reserved.

Programmable RNA recognition and cleavage by CRISPR/Cas9

PubMed Central

O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H.; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A.

2014-01-01

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage1-5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA4-7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition. PMID:25274302

Atomic structure and bonding of the interfacial bilayer between Au nanoparticles and epitaxially regrown MgAl{sub 2}O{sub 4} substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Guo-zhen; Canadian Centre of Electron Microscopy and Department of Materials Science and Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1; Majdi, Tahereh

2014-12-08

A unique metal/oxide interfacial bilayer formed between Au nanoparticles and MgAl{sub 2}O{sub 4} substrates following thermal treatment is reported. Associated with the formation of the bilayer was the onset of an abnormal epitaxial growth of the substrate under the nanoparticle. According to the redistribution of atoms and the changes of their electronic structure probed across the interface by a transmission electron microscopy, we suggest two possible atomic models of the interfacial bilayer.

DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

PubMed

Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

2018-01-01

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis.

PubMed

Juettner, Norbert E; Schmelz, Stefan; Bogen, Jan P; Happel, Dominic; Fessner, Wolf-Dieter; Pfeifer, Felicitas; Fuchsbauer, Hans-Lothar; Scrima, Andrea

2018-05-01

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI p ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI p exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPI p accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI p for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications. © 2018 The Protein Society.

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

PubMed

Gadda, Giovanni; Powell, Nichole L N; Menon, Prashanthi

2004-10-15

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.

Emotion recognition impairment and apathy after subthalamic nucleus stimulation in Parkinson's disease have separate neural substrates.

PubMed

Drapier, D; Péron, J; Leray, E; Sauleau, P; Biseul, I; Drapier, S; Le Jeune, F; Travers, D; Bourguignon, A; Haegelen, C; Millet, B; Vérin, M

2008-09-01

To test the hypothesis that emotion recognition and apathy share the same functional circuit involving the subthalamic nucleus (STN). A consecutive series of 17 patients with advanced Parkinson's disease (PD) was assessed 3 months before (M-3) and 3 months (M+3) after STN deep brain stimulation (DBS). Mean (+/-S.D.) age at surgery was 56.9 (8.7) years. Mean disease duration at surgery was 11.8 (2.6) years. Apathy was measured using the Apathy Evaluation Scale (AES) at both M-3 and M3. Patients were also assessed using a computerised paradigm of facial emotion recognition [Ekman, P., & Friesen, W. V. (1976). Pictures of facial affect. Palo Alto: Consulting Psychologist Press] before and after STN DBS. Prior to this, the Benton Facial Recognition Test was used to check that the ability to perceive faces was intact. Apathy had significantly worsened at M3 (42.5+/-8.9, p=0.006) after STN-DBS, in relation to the preoperative assessment (37.2+/-5.5). There was also a significant reduction in recognition percentages for facial expressions of fear (43.1%+/-22.9 vs. 61.6%+/-21.4, p=0.022) and sadness (52.7%+/-19.1 vs. 67.6%+/-22.8, p=0.031) after STN DBS. However, the postoperative worsening of apathy and emotion recognition impairment were not correlated. Our results confirm that the STN is involved in both the apathy and emotion recognition networks. However, the absence of any correlation between apathy and emotion recognition impairment suggests that the worsening of apathy following surgery could not be explained by a lack of facial emotion recognition and that its behavioural and cognitive components should therefore also be taken into consideration.

Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

PubMed Central

Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

2016-01-01

General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. PMID:27367672

Modular architecture of eukaryotic RNase P and RNase MRP revealed by electron microscopy

PubMed Central

Hipp, Katharina; Galani, Kyriaki; Batisse, Claire; Prinz, Simone; Böttcher, Bettina

2012-01-01

Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined. Using electron microscopy and image processing we show that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules. Such features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence. These are also the sites of greatest difference between RNase P and RNase MRP, highlighting the importance of the adaptation of this region to the different substrates. PMID:22167472

Observing cellulose biosynthesis and membrane translocation in crystallo

PubMed Central

Morgan, Jacob L.W.; McNamara, Joshua T.; Fischer, Michael; Rich, Jamie; Chen, Hong-Ming; Withers, Stephen G.; Zimmer, Jochen

2016-01-01

Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. In crystallo enzymology with the catalytically-active bacterial cellulose synthase BcsA-B complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a “finger helix” that contacts the polymer's terminal glucose. Cooperating with BcsA's gating loop, the finger helix moves ‘up’ and ‘down’ in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA’s transmembrane channel. This mechanism is validated experimentally by tethering BcsA's finger helix, which inhibits polymer translocation but not elongation. PMID:26958837

An allosteric conduit facilitates dynamic multisite substrate recognition by the SCFCdc4 ubiquitin ligase

NASA Astrophysics Data System (ADS)

Csizmok, Veronika; Orlicky, Stephen; Cheng, Jing; Song, Jianhui; Bah, Alaji; Delgoshaie, Neda; Lin, Hong; Mittag, Tanja; Sicheri, Frank; Chan, Hue Sun; Tyers, Mike; Forman-Kay, Julie D.

2017-01-01

The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.

Structural basis of substrate discrimination and integrin binding by autotaxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos

2013-09-25

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates.more » We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.« less

Personal glucose meters for detection and quantification of a broad range of analytes

DOEpatents

Lu, Yi; Xiang, Yu

2015-02-03

A general methodology for the development of highly sensitive and selective sensors that can achieve portable, low-cost and quantitative detection of a broad range of targets using only a personal glucose meter (PGM) is disclosed. The method uses recognition molecules that are specific for a target agent, enzymes that can convert an enzyme substrate into glucose, and PGM. Also provided are sensors, which can include a solid support to which is attached a recognition molecule that permits detection of a target agent, wherein the recognition molecule specifically binds to the target agent in the presence of the target agent but not significantly to other agents as well as an enzyme that can catalyze the conversion of a substance into glucose, wherein the enzyme is attached directly or indirectly to the recognition molecule, and wherein in the presence of the target agent the enzyme can convert the substance into glucose. The disclosed sensors can be part of a lateral flow device. Methods of using such sensors for detecting target agents are also provided.

A multidisciplinary study of earth resources imagery of Australia, Antarctica and Papua, New Guinea

NASA Technical Reports Server (NTRS)

Fisher, N. H. (Principal Investigator)

1975-01-01

The author has identified the following significant results. A thirteen category recognition map was prepared, showing forest, water, grassland, and exposed rock types. Preliminary assessment of classification accuracies showed that water, forest, meadow, and Niobrara shale were the most accurately mapped classes. Unsatisfactory results, were obtained in an attempt to discrimate sparse forest cover over different substrates. As base elevation varied from 7,000 to 13,000 ft, with an atmospheric visibility of 48 km, no changes in water and forest recognition were observed. Granodiorite recognition accuracy decreased monotonically as base elevation increased, even though the training set location was at 10,000 ft elevation. For snow varying in base elevation from 9400 to 8420 ft, recognition decreases from 99% at the 9400 ft training set elevation to 88% at 8420 ft. Calculations of expected radiance at the ERTS sensor from snow reflectance measured at the site and from Turner model calculations of irradiance, transmission and path radiance, reveal that snow signals should not be clipped, assuming that calculations and ERTS calibration constants were correct.

From Perception to Recognition Memory: Time Course and Lateralization of Neural Substrates of Word and Abstract Picture Processing

ERIC Educational Resources Information Center

Maillard, Louis; Barbeau, Emmanuel J.; Baumann, Cedric; Koessler, Laurent; Benar, Christian; Chauvel, Patrick; Liegeois-Chauvel, Catherine

2011-01-01

Through study of clinical cases with brain lesions as well as neuroimaging studies of cognitive processing of words and pictures, it has been established that material-specific hemispheric specialization exists. It remains however unclear whether such specialization holds true for all processes involved in complex tasks, such as recognition…

From containers to catalysts: supramolecular catalysis within cucurbiturils.

PubMed

Pemberton, Barry C; Raghunathan, Ramya; Volla, Sabine; Sivaguru, Jayaraman

2012-09-24

Cucurbiturils are a family of molecular container compounds with superior molecular recognition properties. The use of cucurbiturils for supramolecular catalysis is highlighted in this concept. Both photochemical reactions as well as thermal transformations are reviewed with an eye towards tailoring substrates for supramolecular catalysis mediated by cucurbiturils. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Atomic resolution x-ray structure of the substrate recognition domain of higher plant rubisco activase

USDA-ARS?s Scientific Manuscript database

The rapid release of tight-binding inhibitors from dead-end Rubisco complexes requires the activity of Rubisco activase, an AAA+ ATPase that utilizes chemo-mechanical energy to catalyze the reactivation of Rubisco. Activase is thought to play a central role in coordinating the rate of CO2 fixation w...

Conformational Plasticity of an Enzyme during Catalysis: Intricate Coupling between Cyclophilin A Dynamics and Substrate Turnover

PubMed Central

McGowan, Lauren C.; Hamelberg, Donald

2013-01-01

Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. PMID:23332074

Conformational plasticity of an enzyme during catalysis: intricate coupling between cyclophilin A dynamics and substrate turnover.

PubMed

McGowan, Lauren C; Hamelberg, Donald

2013-01-08

Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

PrenDB, a Substrate Prediction Database to Enable Biocatalytic Use of Prenyltransferases.

PubMed

Gunera, Jakub; Kindinger, Florian; Li, Shu-Ming; Kolb, Peter

2017-03-10

Prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily catalyze the attachment of prenyl or prenyl-like moieties to diverse acceptor compounds. These acceptor molecules are generally aromatic in nature and mostly indole or indole-like. Their catalytic transformation represents a major skeletal diversification step in the biosynthesis of secondary metabolites, including the indole alkaloids. DMATS enzymes thus contribute significantly to the biological and pharmacological diversity of small molecule metabolites. Understanding the substrate specificity of these enzymes could create opportunities for their biocatalytic use in preparing complex synthetic scaffolds. However, there has been no framework to achieve this in a rational way. Here, we report a chemoinformatic pipeline to enable prenyltransferase substrate prediction. We systematically catalogued 32 unique prenyltransferases and 167 unique substrates to create possible reaction matrices and compiled these data into a browsable database named PrenDB. We then used a newly developed algorithm based on molecular fragmentation to automatically extract reactive chemical epitopes. The analysis of the collected data sheds light on the thus far explored substrate space of DMATS enzymes. To assess the predictive performance of our virtual reaction extraction tool, 38 potential substrates were tested as prenyl acceptors in assays with three prenyltransferases, and we were able to detect turnover in >55% of the cases. The database, PrenDB (www.kolblab.org/prendb.php), enables the prediction of potential substrates for chemoenzymatic synthesis through substructure similarity and virtual chemical transformation techniques. It aims at making prenyltransferases and their highly regio- and stereoselective reactions accessible to the research community for integration in synthetic work flows. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Capturing specific abilities as a window into human individuality: the example of face recognition.

PubMed

Wilmer, Jeremy B; Germine, Laura; Chabris, Christopher F; Chatterjee, Garga; Gerbasi, Margaret; Nakayama, Ken

2012-01-01

Proper characterization of each individual's unique pattern of strengths and weaknesses requires good measures of diverse abilities. Here, we advocate combining our growing understanding of neural and cognitive mechanisms with modern psychometric methods in a renewed effort to capture human individuality through a consideration of specific abilities. We articulate five criteria for the isolation and measurement of specific abilities, then apply these criteria to face recognition. We cleanly dissociate face recognition from more general visual and verbal recognition. This dissociation stretches across ability as well as disability, suggesting that specific developmental face recognition deficits are a special case of a broader specificity that spans the entire spectrum of human face recognition performance. Item-by-item results from 1,471 web-tested participants, included as supplementary information, fuel item analyses, validation, norming, and item response theory (IRT) analyses of our three tests: (a) the widely used Cambridge Face Memory Test (CFMT); (b) an Abstract Art Memory Test (AAMT), and (c) a Verbal Paired-Associates Memory Test (VPMT). The availability of this data set provides a solid foundation for interpreting future scores on these tests. We argue that the allied fields of experimental psychology, cognitive neuroscience, and vision science could fuel the discovery of additional specific abilities to add to face recognition, thereby providing new perspectives on human individuality.

Capacities for theory of mind, metacognition, and neurocognitive function are independently related to emotional recognition in schizophrenia.

PubMed

Lysaker, Paul H; Leonhardt, Bethany L; Brüne, Martin; Buck, Kelly D; James, Alison; Vohs, Jenifer; Francis, Michael; Hamm, Jay A; Salvatore, Giampaolo; Ringer, Jamie M; Dimaggio, Giancarlo

2014-09-30

While many with schizophrenia spectrum disorders experience difficulties understanding the feelings of others, little is known about the psychological antecedents of these deficits. To explore these issues we examined whether deficits in mental state decoding, mental state reasoning and metacognitive capacity predict performance on an emotion recognition task. Participants were 115 adults with a schizophrenia spectrum disorder and 58 adults with substance use disorders but no history of a diagnosis of psychosis who completed the Eyes and Hinting Test. Metacognitive capacity was assessed using the Metacognitive Assessment Scale Abbreviated and emotion recognition was assessed using the Bell Lysaker Emotion Recognition Test. Results revealed that the schizophrenia patients performed more poorly than controls on tests of emotion recognition, mental state decoding, mental state reasoning and metacognition. Lesser capacities for mental state decoding, mental state reasoning and metacognition were all uniquely related emotion recognition within the schizophrenia group even after controlling for neurocognition and symptoms in a stepwise multiple regression. Results suggest that deficits in emotion recognition in schizophrenia may partly result from a combination of impairments in the ability to judge the cognitive and affective states of others and difficulties forming complex representations of self and others. Published by Elsevier Ireland Ltd.

Method for selective immobilization of macromolecules on self assembled monolayer surfaces

DOEpatents

Laskin, Julia [Richland, WA; Wang, Peng [Billerica, MA

2011-11-29

Disclosed is a method for selective chemical binding and immobilization of macromolecules on solid supports in conjunction with self-assembled monolayer (SAM) surfaces. Immobilization involves selective binding of peptides and other macromolecules to SAM surfaces using reactive landing (RL) of mass-selected, gas phase ions. SAM surfaces provide a simple and convenient platform for tailoring chemical properties of a variety of substrates. The invention finds applications in biochemistry ranging from characterization of molecular recognition events at the amino acid level and identification of biologically active motifs in proteins, to development of novel biosensors and substrates for stimulated protein and cell adhesion.

Brain correlates of musical and facial emotion recognition: evidence from the dementias.

PubMed

Hsieh, S; Hornberger, M; Piguet, O; Hodges, J R

2012-07-01

The recognition of facial expressions of emotion is impaired in semantic dementia (SD) and is associated with right-sided brain atrophy in areas known to be involved in emotion processing, notably the amygdala. Whether patients with SD also experience difficulty recognizing emotions conveyed by other media, such as music, is unclear. Prior studies have used excerpts of known music from classical or film repertoire but not unfamiliar melodies designed to convey distinct emotions. Patients with SD (n = 11), Alzheimer's disease (n = 12) and healthy control participants (n = 20) underwent tests of emotion recognition in two modalities: unfamiliar musical tunes and unknown faces as well as volumetric MRI. Patients with SD were most impaired with the recognition of facial and musical emotions, particularly for negative emotions. Voxel-based morphometry showed that the labelling of emotions, regardless of modality, correlated with the degree of atrophy in the right temporal pole, amygdala and insula. The recognition of musical (but not facial) emotions was also associated with atrophy of the left anterior and inferior temporal lobe, which overlapped with regions correlating with standardized measures of verbal semantic memory. These findings highlight the common neural substrates supporting the processing of emotions by facial and musical stimuli but also indicate that the recognition of emotions from music draws upon brain regions that are associated with semantics in language. Copyright © 2012 Elsevier Ltd. All rights reserved.

Methods and apparatus for non-acoustic speech characterization and recognition

DOEpatents

Holzrichter, John F.

1999-01-01

By simultaneously recording EM wave reflections and acoustic speech information, the positions and velocities of the speech organs as speech is articulated can be defined for each acoustic speech unit. Well defined time frames and feature vectors describing the speech, to the degree required, can be formed. Such feature vectors can uniquely characterize the speech unit being articulated each time frame. The onset of speech, rejection of external noise, vocalized pitch periods, articulator conditions, accurate timing, the identification of the speaker, acoustic speech unit recognition, and organ mechanical parameters can be determined.

Methods and apparatus for non-acoustic speech characterization and recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holzrichter, J.F.

By simultaneously recording EM wave reflections and acoustic speech information, the positions and velocities of the speech organs as speech is articulated can be defined for each acoustic speech unit. Well defined time frames and feature vectors describing the speech, to the degree required, can be formed. Such feature vectors can uniquely characterize the speech unit being articulated each time frame. The onset of speech, rejection of external noise, vocalized pitch periods, articulator conditions, accurate timing, the identification of the speaker, acoustic speech unit recognition, and organ mechanical parameters can be determined.

Multi-font printed Mongolian document recognition system

NASA Astrophysics Data System (ADS)

Peng, Liangrui; Liu, Changsong; Ding, Xiaoqing; Wang, Hua; Jin, Jianming

2009-01-01

Mongolian is one of the major ethnic languages in China. Large amount of Mongolian printed documents need to be digitized in digital library and various applications. Traditional Mongolian script has unique writing style and multi-font-type variations, which bring challenges to Mongolian OCR research. As traditional Mongolian script has some characteristics, for example, one character may be part of another character, we define the character set for recognition according to the segmented components, and the components are combined into characters by rule-based post-processing module. For character recognition, a method based on visual directional feature and multi-level classifiers is presented. For character segmentation, a scheme is used to find the segmentation point by analyzing the properties of projection and connected components. As Mongolian has different font-types which are categorized into two major groups, the parameter of segmentation is adjusted for each group. A font-type classification method for the two font-type group is introduced. For recognition of Mongolian text mixed with Chinese and English, language identification and relevant character recognition kernels are integrated. Experiments show that the presented methods are effective. The text recognition rate is 96.9% on the test samples from practical documents with multi-font-types and mixed scripts.

Concept recognition for extracting protein interaction relations from biomedical text

PubMed Central

Baumgartner, William A; Lu, Zhiyong; Johnson, Helen L; Caporaso, J Gregory; Paquette, Jesse; Lindemann, Anna; White, Elizabeth K; Medvedeva, Olga; Cohen, K Bretonnel; Hunter, Lawrence

2008-01-01

Background: Reliable information extraction applications have been a long sought goal of the biomedical text mining community, a goal that if reached would provide valuable tools to benchside biologists in their increasingly difficult task of assimilating the knowledge contained in the biomedical literature. We present an integrated approach to concept recognition in biomedical text. Concept recognition provides key information that has been largely missing from previous biomedical information extraction efforts, namely direct links to well defined knowledge resources that explicitly cement the concept's semantics. The BioCreative II tasks discussed in this special issue have provided a unique opportunity to demonstrate the effectiveness of concept recognition in the field of biomedical language processing. Results: Through the modular construction of a protein interaction relation extraction system, we present several use cases of concept recognition in biomedical text, and relate these use cases to potential uses by the benchside biologist. Conclusion: Current information extraction technologies are approaching performance standards at which concept recognition can begin to deliver high quality data to the benchside biologist. Our system is available as part of the BioCreative Meta-Server project and on the internet . PMID:18834500

Wavelet Types Comparison for Extracting Iris Feature Based on Energy Compaction

NASA Astrophysics Data System (ADS)

Rizal Isnanto, R.

2015-06-01

Human iris has a very unique pattern which is possible to be used as a biometric recognition. To identify texture in an image, texture analysis method can be used. One of method is wavelet that extract the image feature based on energy. Wavelet transforms used are Haar, Daubechies, Coiflets, Symlets, and Biorthogonal. In the research, iris recognition based on five mentioned wavelets was done and then comparison analysis was conducted for which some conclusions taken. Some steps have to be done in the research. First, the iris image is segmented from eye image then enhanced with histogram equalization. The features obtained is energy value. The next step is recognition using normalized Euclidean distance. Comparison analysis is done based on recognition rate percentage with two samples stored in database for reference images. After finding the recognition rate, some tests are conducted using Energy Compaction for all five types of wavelets above. As the result, the highest recognition rate is achieved using Haar, whereas for coefficients cutting for C(i) < 0.1, Haar wavelet has a highest percentage, therefore the retention rate or significan coefficient retained for Haaris lower than other wavelet types (db5, coif3, sym4, and bior2.4)

The review and results of different methods for facial recognition

NASA Astrophysics Data System (ADS)

Le, Yifan

2017-09-01

In recent years, facial recognition draws much attention due to its wide potential applications. As a unique technology in Biometric Identification, facial recognition represents a significant improvement since it could be operated without cooperation of people under detection. Hence, facial recognition will be taken into defense system, medical detection, human behavior understanding, etc. Several theories and methods have been established to make progress in facial recognition: (1) A novel two-stage facial landmark localization method is proposed which has more accurate facial localization effect under specific database; (2) A statistical face frontalization method is proposed which outperforms state-of-the-art methods for face landmark localization; (3) It proposes a general facial landmark detection algorithm to handle images with severe occlusion and images with large head poses; (4) There are three methods proposed on Face Alignment including shape augmented regression method, pose-indexed based multi-view method and a learning based method via regressing local binary features. The aim of this paper is to analyze previous work of different aspects in facial recognition, focusing on concrete method and performance under various databases. In addition, some improvement measures and suggestions in potential applications will be put forward.

Cognitive object recognition system (CORS)

NASA Astrophysics Data System (ADS)

Raju, Chaitanya; Varadarajan, Karthik Mahesh; Krishnamurthi, Niyant; Xu, Shuli; Biederman, Irving; Kelley, Troy

2010-04-01

We have developed a framework, Cognitive Object Recognition System (CORS), inspired by current neurocomputational models and psychophysical research in which multiple recognition algorithms (shape based geometric primitives, 'geons,' and non-geometric feature-based algorithms) are integrated to provide a comprehensive solution to object recognition and landmarking. Objects are defined as a combination of geons, corresponding to their simple parts, and the relations among the parts. However, those objects that are not easily decomposable into geons, such as bushes and trees, are recognized by CORS using "feature-based" algorithms. The unique interaction between these algorithms is a novel approach that combines the effectiveness of both algorithms and takes us closer to a generalized approach to object recognition. CORS allows recognition of objects through a larger range of poses using geometric primitives and performs well under heavy occlusion - about 35% of object surface is sufficient. Furthermore, geon composition of an object allows image understanding and reasoning even with novel objects. With reliable landmarking capability, the system improves vision-based robot navigation in GPS-denied environments. Feasibility of the CORS system was demonstrated with real stereo images captured from a Pioneer robot. The system can currently identify doors, door handles, staircases, trashcans and other relevant landmarks in the indoor environment.

Substrate influence on the interlayer electron-phonon couplings in fullerene films probed with doubly-resonant SFG spectroscopy.

PubMed

Elsenbeck, Dennis; Das, Sushanta K; Velarde, Luis

2017-07-19

We present doubly-resonant sum frequency generation (DR-SFG) spectra of fullerene thin films on metallic and dielectric substrates as a way to investigate the interplay between nuclear and electronic coupling at buried interfaces. Modal and substrate selectivity in the electronic enhancement of the C 60 vibrational signatures is demonstrated for excitation wavelengths spanning the visible range. While the SFG response of the totally symmetric A g (2) mode of fullerene is distinctly coupled to the optically allowed electronic transition corresponding to the HOMO-LUMO+1 of C 60 (ca. 2.6 eV), the T 1u (4) vibrational mode appears to be coupled to a symmetry-forbidden HOMO-LUMO transition at lower energies (ca. 2.0 eV). For dielectric substrates, the DR-SFG intensity of the T 1u (4) mode shows lack of enhancement for upconversion wavelengths off-resonance with the optically-dark LUMO. However, the T 1u (4) mode shows a unique coupling to an intermediate state (∼2.4 eV) only for the fullerene films on the gold substrate. We attribute this coupling to unique interactions at the buried C 60 /gold interface. These results demonstrate the occurrence of clear electron-phonon couplings at the C 60 /substrate interfaces and shed light on the impact of these couplings on the optical response of electronically excited fullerene. This coupling may influence charge and energy transport in organic electronic devices mediated by vibrational motions. We also demonstrate a potential use of this added selectivity in chemical imaging.

Substrate Type and Free Ammonia Determine Bacterial Community Structure in Full-Scale Mesophilic Anaerobic Digesters Treating Cattle or Swine Manure.

PubMed

Li, Jiabao; Rui, Junpeng; Yao, Minjie; Zhang, Shiheng; Yan, Xuefeng; Wang, Yuanpeng; Yan, Zhiying; Li, Xiangzhen

2015-01-01

The microbial-mediated anaerobic digestion (AD) process represents an efficient biological process for the treatment of organic waste along with biogas harvest. Currently, the key factors structuring bacterial communities and the potential core and unique bacterial populations in manure anaerobic digesters are not completely elucidated yet. In this study, we collected sludge samples from 20 full-scale anaerobic digesters treating cattle or swine manure, and investigated the variations of bacterial community compositions using high-throughput 16S rRNA amplicon sequencing. Clustering and correlation analysis suggested that substrate type and free ammonia (FA) play key roles in determining the bacterial community structure. The COD: [Formula: see text] (C:N) ratio of substrate and FA were the most important available operational parameters correlating to the bacterial communities in cattle and swine manure digesters, respectively. The bacterial populations in all of the digesters were dominated by phylum Firmicutes, followed by Bacteroidetes, Proteobacteria and Chloroflexi. Increased FA content selected Firmicutes, suggesting that they probably play more important roles under high FA content. Syntrophic metabolism by Proteobacteria, Chloroflexi, Synergistetes and Planctomycetes are likely inhibited when FA content is high. Despite the different manure substrates, operational conditions and geographical locations of digesters, core bacterial communities were identified. The core communities were best characterized by phylum Firmicutes, wherein Clostridium predominated overwhelmingly. Substrate-unique and abundant communities may reflect the properties of manure substrate and operational conditions. These findings extend our current understanding of the bacterial assembly in full-scale manure anaerobic digesters.

Substrate Type and Free Ammonia Determine Bacterial Community Structure in Full-Scale Mesophilic Anaerobic Digesters Treating Cattle or Swine Manure

PubMed Central

Li, Jiabao; Rui, Junpeng; Yao, Minjie; Zhang, Shiheng; Yan, Xuefeng; Wang, Yuanpeng; Yan, Zhiying; Li, Xiangzhen

2015-01-01

The microbial-mediated anaerobic digestion (AD) process represents an efficient biological process for the treatment of organic waste along with biogas harvest. Currently, the key factors structuring bacterial communities and the potential core and unique bacterial populations in manure anaerobic digesters are not completely elucidated yet. In this study, we collected sludge samples from 20 full-scale anaerobic digesters treating cattle or swine manure, and investigated the variations of bacterial community compositions using high-throughput 16S rRNA amplicon sequencing. Clustering and correlation analysis suggested that substrate type and free ammonia (FA) play key roles in determining the bacterial community structure. The COD: NH4+-N (C:N) ratio of substrate and FA were the most important available operational parameters correlating to the bacterial communities in cattle and swine manure digesters, respectively. The bacterial populations in all of the digesters were dominated by phylum Firmicutes, followed by Bacteroidetes, Proteobacteria and Chloroflexi. Increased FA content selected Firmicutes, suggesting that they probably play more important roles under high FA content. Syntrophic metabolism by Proteobacteria, Chloroflexi, Synergistetes and Planctomycetes are likely inhibited when FA content is high. Despite the different manure substrates, operational conditions and geographical locations of digesters, core bacterial communities were identified. The core communities were best characterized by phylum Firmicutes, wherein Clostridium predominated overwhelmingly. Substrate-unique and abundant communities may reflect the properties of manure substrate and operational conditions. These findings extend our current understanding of the bacterial assembly in full-scale manure anaerobic digesters. PMID:26648921

Medial Prefrontal Cortex and Dorsomedial Striatum Are Necessary for the Trial-Unique, Delayed Nonmatching-to-Location (TUNL) Task in Rats: Role of NMDA Receptors

ERIC Educational Resources Information Center

Davies, Don A.; Hurtubise, Jessica L.; Greba, Quentin; Howland, John G.

2017-01-01

The trial-unique, delayed nonmatching-to-location (TUNL) task is a recently developed behavioral task that measures spatial working memory and a form of pattern separation in touchscreen-equipped operant conditioning chambers. Limited information exists regarding the neurotransmitters and neural substrates involved in the task. The present…

Simple thermal to thermal face verification method based on local texture descriptors

NASA Astrophysics Data System (ADS)

Grudzien, A.; Palka, Norbert; Kowalski, M.

2017-08-01

Biometrics is a science that studies and analyzes physical structure of a human body and behaviour of people. Biometrics found many applications ranging from border control systems, forensics systems for criminal investigations to systems for access control. Unique identifiers, also referred to as modalities are used to distinguish individuals. One of the most common and natural human identifiers is a face. As a result of decades of investigations, face recognition achieved high level of maturity, however recognition in visible spectrum is still challenging due to illumination aspects or new ways of spoofing. One of the alternatives is recognition of face in different parts of light spectrum, e.g. in infrared spectrum. Thermal infrared offer new possibilities for human recognition due to its specific properties as well as mature equipment. In this paper we present the scheme of subject's verification methodology by using facial images in thermal range. The study is focused on the local feature extraction methods and on the similarity metrics. We present comparison of two local texture-based descriptors for thermal 1-to-1 face recognition.

Cross-modal working memory binding and word recognition skills: how specific is the link?

PubMed

Wang, Shinmin; Allen, Richard J

2018-04-01

Recent research has suggested that the creation of temporary bound representations of information from different sources within working memory uniquely relates to word recognition abilities in school-age children. However, it is unclear to what extent this link is attributable specifically to the binding ability for cross-modal information. This study examined the performance of Grade 3 (8-9 years old) children on binding tasks requiring either temporary association formation of two visual items (i.e., within-modal binding) or pairs of visually presented abstract shapes and auditorily presented nonwords (i.e., cross-modal binding). Children's word recognition skills were related to performance on the cross-modal binding task but not on the within-modal binding task. Further regression models showed that cross-modal binding memory was a significant predictor of word recognition when memory for its constituent elements, general abilities, and crucially, within-modal binding memory were taken into account. These findings may suggest a specific link between the ability to bind information across modalities within working memory and word recognition skills.

Darwin revisited: The vagus nerve is a causal element in controlling recognition of other's emotions.

PubMed

Colzato, Lorenza S; Sellaro, Roberta; Beste, Christian

2017-07-01

Charles Darwin proposed that via the vagus nerve, the tenth cranial nerve, emotional facial expressions are evolved, adaptive and serve a crucial communicative function. In line with this idea, the later-developed polyvagal theory assumes that the vagus nerve is the key phylogenetic substrate that regulates emotional and social behavior. The polyvagal theory assumes that optimal social interaction, which includes the recognition of emotion in faces, is modulated by the vagus nerve. So far, in humans, it has not yet been demonstrated that the vagus plays a causal role in emotion recognition. To investigate this we employed transcutaneous vagus nerve stimulation (tVNS), a novel non-invasive brain stimulation technique that modulates brain activity via bottom-up mechanisms. A sham/placebo-controlled, randomized cross-over within-subjects design was used to infer a causal relation between the stimulated vagus nerve and the related ability to recognize emotions as indexed by the Reading the Mind in the Eyes Test in 38 healthy young volunteers. Active tVNS, compared to sham stimulation, enhanced emotion recognition for easy items, suggesting that it promoted the ability to decode salient social cues. Our results confirm that the vagus nerve is causally involved in emotion recognition, supporting Darwin's argumentation. Copyright © 2017 Elsevier Ltd. All rights reserved.

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

PubMed

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

Iris Cryptography for Security Purpose

NASA Astrophysics Data System (ADS)

Ajith, Srighakollapu; Balaji Ganesh Kumar, M.; Latha, S.; Samiappan, Dhanalakshmi; Muthu, P.

2018-04-01

In today's world, the security became the major issue to every human being. A major issue is hacking as hackers are everywhere, as the technology was developed still there are many issues where the technology fails to meet the security. Engineers, scientists were discovering the new products for security purpose as biometrics sensors like face recognition, pattern recognition, gesture recognition, voice authentication etcetera. But these devices fail to reach the expected results. In this work, we are going to present an approach to generate a unique secure key using the iris template. Here the iris templates are processed using the well-defined processing techniques. Using the encryption and decryption process they are stored, traversed and utilized. As of the work, we can conclude that the iris cryptography gives us the expected results for securing the data from eavesdroppers.

Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursormore » protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.« less

Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

DOE PAGES

Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying; ...

2015-07-14

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursormore » protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.« less

The Role of the Telomere End Protection Complex in Telomere Main

DTIC Science & Technology

2003-06-01

identification of putative substrates of ATM kinase family members. J Biol Chem, 1999. 274(53): p. 37538-43. 9. Lei, M., E.R. Podell , P. Baumann, and T.R. Cech...DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA. Nature, 2003. 426(6963): p. 198-203. 10. Lei, M., E.R. Podell

Investigation of Dynamic Algorithms for Pattern Recognition Identified in Cerebral Cortex

DTIC Science & Technology

1991-12-02

oscillatory and possibly chaotic activity forin the actual cortical substrate of the diverse sensory, motor, and cognitive operations now studied in...September Neural Information Processing Systems - Natural and Synthetic, Denver, Colo., November 1989 U.C. San Diego, Cognitive Science Dept...Baird. Biologically applied neural networks may foster the co-evolution of neurobiology and cognitive psychology. Brain and Behavioral Sciences, 37

Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

PubMed

Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

2015-12-11

Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

PubMed Central

Butler, Christopher F.; Peet, Caroline; Mason, Amy E.; Voice, Michael W.; Leys, David; Munro, Andrew W.

2013-01-01

Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

PubMed

Galiullina, Raisa A; Kasperkiewicz, Paulina; Chichkova, Nina V; Szalek, Aleksandra; Serebryakova, Marina V; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B

2015-10-09

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Electrophoretic-deposited CNT/MnO2 composites for high-power electrochemical energy storage/conversion applications

NASA Astrophysics Data System (ADS)

Xiao, Wei; Xia, Hui; Fuh, Jerry Y. H.; Lu, Li

2010-05-01

CNT/MnO2 (birnessite-type) composite films have been successfully deposited on Ni-foil substrate via electrophoretic deposition (EPD). The unique EPD CNT/MnO2 composite film electrode shows enhanced electrical conductivity, good contact between composite films and the substrate and open porous structure, which makes the EPD composite films a promising electrode for high-power supercapacitors and lithium ion batteries.

Spatial Object Recognition Enables Endogenous LTD that Curtails LTP in the Mouse Hippocampus

PubMed Central

Goh, Jinzhong Jeremy

2013-01-01

Although synaptic plasticity is believed to comprise the cellular substrate for learning and memory, limited direct evidence exists that hippocampus-dependent learning actually triggers synaptic plasticity. It is likely, however, that long-term potentiation (LTP) works in concert with its counterpart, long-term depression (LTD) in the creation of spatial memory. It has been reported in rats that weak synaptic plasticity is facilitated into persistent plasticity if afferent stimulation is coupled with a novel spatial learning event. It is not known if this phenomenon also occurs in other species. We recorded from the hippocampal CA1 of freely behaving mice and observed that novel spatial learning triggers endogenous LTD. Specifically, we observed that LTD is enabled when test-pulse afferent stimulation is given during the learning of object constellations or during a spatial object recognition task. Intriguingly, LTP is significantly impaired by the same tasks, suggesting that LTD is the main cellular substrate for this type of learning. These data indicate that learning-facilitated plasticity is not exclusive to rats and that spatial learning leads to endogenous LTD in the hippocampus, suggesting an important role for this type of synaptic plasticity in the creation of hippocampus-dependent memory. PMID:22510536

Structure and sequence based functional annotation of Zika virus NS2b protein: Computational insights.

PubMed

Aguilera-Pesantes, Daniel; Méndez, Miguel A

2017-10-28

While Zika virus (ZIKV) outbreaks are a growing concern for global health, a deep understanding about the virus is lacking. Here we report a contribution to the basic science on the virus- a detailed computational analysis of the non structural protein NS2b. This protein acts as a cofactor for the NS3 protease (NS3Pro) domain that is important on the viral life cycle, and is an interesting target for drug development. We found that ZIKV NS2b cofactor is highly similar to other virus within the Flavivirus genus, especially to West Nile Virus, suggesting that it is completely necessary for the protease complex activity. Furthermore, the ZIKV NS2b has an important role to the function and stability of the ZIKV NS3 protease domain even when presents a low conservation score. In addition, ZIKV NS2b is mostly rigid, which could imply a non dynamic nature in substrate recognition. Finally, by performing a computational alanine scanning mutagenesis, we found that residues Gly 52 and Asp 83 in the NS2b could be important in substrate recognition. Copyright © 2017 Elsevier Inc. All rights reserved.

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III*

PubMed Central

Kuznetsov, Nikita A.; Kladova, Olga A.; Kuznetsova, Alexandra A.; Ishchenko, Alexander A.; Saparbaev, Murat K.; Zharkov, Dmitry O.; Fedorova, Olga S.

2015-01-01

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3′-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln41 and Leu81 as DNA lesion sensors. PMID:25869130

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis.

PubMed

Shemesh, Jonathan; Ben Arye, Tom; Avesar, Jonathan; Kang, Joo H; Fine, Amir; Super, Michael; Meller, Amit; Ingber, Donald E; Levenberg, Shulamit

2014-08-05

Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required.

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

PubMed Central

Shemesh, Jonathan; Ben Arye, Tom; Avesar, Jonathan; Kang, Joo H.; Fine, Amir; Super, Michael; Meller, Amit; Ingber, Donald E.; Levenberg, Shulamit

2014-01-01

Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required. PMID:25053808

Placing a Disrupted Degradation Motif at the C Terminus of Proteasome Substrates Attenuates Degradation without Impairing Ubiquitylation*

PubMed Central

Alfassy, Omri S.; Cohen, Itamar; Reiss, Yuval; Tirosh, Boaz; Ravid, Tommer

2013-01-01

Protein elimination by the ubiquitin-proteasome system requires the presence of a cis-acting degradation signal. Efforts to discern degradation signals of misfolded proteasome substrates thus far revealed a general mechanism whereby the exposure of cryptic hydrophobic motifs provides a degradation determinant. We have previously characterized such a determinant, employing the yeast kinetochore protein Ndc10 as a model substrate. Ndc10 is essentially a stable protein that is rapidly degraded upon exposure of a hydrophobic motif located at the C-terminal region. The degradation motif comprises two distinct and essential elements: DegA, encompassing two amphipathic helices, and DegB, a hydrophobic sequence within the loosely structured C-terminal tail of Ndc10. Here we show that the hydrophobic nature of DegB is irrelevant for the ubiquitylation of substrates containing the Ndc10 degradation motif, but is essential for proteasomal degradation. Mutant DegB, in which the hydrophobic sequence was disrupted, acted as a dominant degradation inhibitory element when expressed at the C-terminal regions of ubiquitin-dependent and -independent substrates of the 26S proteasome. This mutant stabilized substrates in both yeast and mammalian cells, indicative of a modular recognition moiety. The dominant function of the mutant DegB provides a powerful experimental tool for evaluating the physiological implications of stabilization of specific proteasome substrates in intact cells and for studying the associated pathological effects. PMID:23519465

Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Seungil; Chang, Jeanne S.; Griffor, Matt

DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2more » tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.« less

RNA-Catalyzed RNA Ligation on an External RNA Template

NASA Technical Reports Server (NTRS)

McGinness, Kathleen E.; Joyce, Gerald F.

2002-01-01

Variants of the hc ligase ribozyme, which catalyzes ligation of the 3' end of an RNA substrate to the 5' end of the ribozyme, were utilized to evolve a ribozyme that catalyzes ligation reactions on an external RNA template. The evolved ribozyme catalyzes the joining of an oligonucleotide 3'-hydroxyl to the 5'-triphosphate of an RNA hairpin molecule. The ribozyme can also utilize various substrate sequences, demonstrating a largely sequence-independent mechanism for substrate recognition. The ribozyme also carries out the ligation of two oligonucleotides that are bound at adjacent positions on a complementary template. Finally, it catalyzes addition of mononucleoside '5-triphosphates onto the '3 end of an oligonucleotide primer in a template-dependent manner. The development of ribozymes that catalyze polymerase-type reactions contributes to the notion that an RNA world could have existed during the early history of life on Earth.

Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Yong; Karaveg, Khanita; Moremen, Kelley W.

2016-11-17

Asn-linked glycosylation of newly synthesized polypeptides occurs in the endoplasmic reticulum of eukaryotic cells. Glycan structures are trimmed and remodeled as they transit the secretory pathway, and processing intermediates play various roles as ligands for folding chaperones and signals for quality control and intracellular transport. Key steps for the generation of these trimmed intermediates are catalyzed by glycoside hydrolase family 47 (GH47) α-mannosidases that selectively cleave α1,2-linked mannose residues. Despite the sequence and structural similarities among the GH47 enzymes, the molecular basis for residue-specific cleavage remains obscure. The present studies reveal enzyme–substrate complex structures for two related GH47 α-mannosidases andmore » provide insights into how these enzymes recognize the same substrates differently and catalyze the complementary glycan trimming reactions necessary for glycan maturation.« less

Assignment of function to Histidines 260 and 298 by engineering the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase complex; substitutions that lead to acceptance of substrates lacking the 5-carboxyl group.†

PubMed Central

Shim, Da Jeong; Nemeria, Natalia S.; Balakrishnan, Anand; Patel, Hetalben; Song, Jaeyoung; Wang, Junjie; Jordan, Frank; Farinas, Edgardo T.

2011-01-01

The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced by hydrophobic residues of similar molecular volume. To interrogate whether the second component would enable synthesis of acyl-coenzymeA derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the ‘gatekeeper’ for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate, but E2o for 2-oxoglutarate. PMID:21809826

Structure and Function of the 26S Proteasome.

PubMed

Bard, Jared A M; Goodall, Ellen A; Greene, Eric R; Jonsson, Erik; Dong, Ken C; Martin, Andreas

2018-06-20

As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.

Ubiquitin Ligases: Structure, Function, and Regulation.

PubMed

Zheng, Ning; Shabek, Nitzan

2017-06-20

Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.

Substrate effect on the growth of monolayer dendritic MoS2 on LaAlO3 (100) and its electrocatalytic applications

NASA Astrophysics Data System (ADS)

Li, Cong; Zhang, Yu; Ji, Qingqing; Shi, Jianping; Chen, Zhaolong; Zhou, Xiebo; Fang, Qiyi; Zhang, Yanfeng

2016-09-01

In accommodating the rapid development of two-dimensional (2D) nanomaterials, chemical vapor deposition (CVD) has become a powerful tool for their batch production with desirable characteristics, such as high crystal quality, large domain size, and tunable domain shape. The crystallinity and morphology of the growth substrates usually play a crucial role in the CVD synthesis of high-quality monolayer MoS2, a kind of 2D layered material which has ignited huge interest in nanoelectronics, optoelectronics and energy harvesting, etc. Herein, by utilizing a low-pressure chemical vapor deposition (LPCVD) system, we demonstrate a regioselective synthesis of monolayer MoS2 on the corrugated single-crystal LaAlO3 (100) with twin crystal domains induced by the second-order phase transition. Unique dendritic morphologies with tunable nucleation densities were obtained in different regions of the undulated substrate, presenting a strong substrate modulation effect. Interestingly, the exposure of abundant active edge sites along with the rather high nucleation density makes the monolayer dendritic MoS2 a good electrocatalyst for hydrogen evolution reaction (HER), particularly featured by a rather high exchange current density (70.4 μA cm-2). Furthermore, uniform monolayer MoS2 films can also be obtained and transferred to arbitrary substrates. We believe that this work provides a new growth system for the controllable synthesis of 2D layered materials with unique dendritic morphologies, as well as its great application potential in energy conversion and harvesting.

Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter.

PubMed

Barker, E L; Moore, K R; Rakhshan, F; Blakely, R D

1999-06-15

Mutation of a conserved Asp (D98) in the rat serotonin (5HT) transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in 5HT Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of 5HT transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to 5HT for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to 5HT occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters.

Recognition memory is modulated by visual similarity.

PubMed

Yago, Elena; Ishai, Alumit

2006-06-01

We used event-related fMRI to test whether recognition memory depends on visual similarity between familiar prototypes and novel exemplars. Subjects memorized portraits, landscapes, and abstract compositions by six painters with a unique style, and later performed a memory recognition task. The prototypes were presented with new exemplars that were either visually similar or dissimilar. Behaviorally, novel, dissimilar items were detected faster and more accurately. We found activation in a distributed cortical network that included face- and object-selective regions in the visual cortex, where familiar prototypes evoked stronger responses than new exemplars; attention-related regions in parietal cortex, where responses elicited by new exemplars were reduced with decreased similarity to the prototypes; and the hippocampus and memory-related regions in parietal and prefrontal cortices, where stronger responses were evoked by the dissimilar exemplars. Our findings suggest that recognition memory is mediated by classification of novel exemplars as a match or a mismatch, based on their visual similarity to familiar prototypes.

Gabor filter based fingerprint image enhancement

NASA Astrophysics Data System (ADS)

Wang, Jin-Xiang

2013-03-01

Fingerprint recognition technology has become the most reliable biometric technology due to its uniqueness and invariance, which has been most convenient and most reliable technique for personal authentication. The development of Automated Fingerprint Identification System is an urgent need for modern information security. Meanwhile, fingerprint preprocessing algorithm of fingerprint recognition technology has played an important part in Automatic Fingerprint Identification System. This article introduces the general steps in the fingerprint recognition technology, namely the image input, preprocessing, feature recognition, and fingerprint image enhancement. As the key to fingerprint identification technology, fingerprint image enhancement affects the accuracy of the system. It focuses on the characteristics of the fingerprint image, Gabor filters algorithm for fingerprint image enhancement, the theoretical basis of Gabor filters, and demonstration of the filter. The enhancement algorithm for fingerprint image is in the windows XP platform with matlab.65 as a development tool for the demonstration. The result shows that the Gabor filter is effective in fingerprint image enhancement technology.

Neural Substrates of View-Invariant Object Recognition Developed without Experiencing Rotations of the Objects

PubMed Central

Okamura, Jun-ya; Yamaguchi, Reona; Honda, Kazunari; Tanaka, Keiji

2014-01-01

One fails to recognize an unfamiliar object across changes in viewing angle when it must be discriminated from similar distractor objects. View-invariant recognition gradually develops as the viewer repeatedly sees the objects in rotation. It is assumed that different views of each object are associated with one another while their successive appearance is experienced in rotation. However, natural experience of objects also contains ample opportunities to discriminate among objects at each of the multiple viewing angles. Our previous behavioral experiments showed that after experiencing a new set of object stimuli during a task that required only discrimination at each of four viewing angles at 30° intervals, monkeys could recognize the objects across changes in viewing angle up to 60°. By recording activities of neurons from the inferotemporal cortex after various types of preparatory experience, we here found a possible neural substrate for the monkeys' performance. For object sets that the monkeys had experienced during the task that required only discrimination at each of four viewing angles, many inferotemporal neurons showed object selectivity covering multiple views. The degree of view generalization found for these object sets was similar to that found for stimulus sets with which the monkeys had been trained to conduct view-invariant recognition. These results suggest that the experience of discriminating new objects in each of several viewing angles develops the partially view-generalized object selectivity distributed over many neurons in the inferotemporal cortex, which in turn bases the monkeys' emergent capability to discriminate the objects across changes in viewing angle. PMID:25378169

Executive Dysfunction among Children with Reading Comprehension Deficits

ERIC Educational Resources Information Center

Locascio, Gianna; Mahone, E. Mark; Eason, Sarah H.; Cutting, Laurie E.

2010-01-01

Emerging research supports the contribution of executive function (EF) to reading comprehension; however, a unique pattern has not been established for children who demonstrate comprehension difficulties despite average word recognition ability (specific reading comprehension deficit; S-RCD). To identify particular EF components on which children…

Neural Network--OCR/ICR Recognology: Theory and Applications.

ERIC Educational Resources Information Center

Schantz, Herbert F.

1993-01-01

Explains the value of neurocomputing as a unique and effective new technological concept for information processing and optical character recognition. Comparisons are made to digital computing and examples of applications such as recognizing handprinted characters are addressed. Products available from various companies are described. (Contains…

Vertex Space Analysis for Model-Based Target Recognition.

DTIC Science & Technology

1996-08-01

performed in our unique invariant representation, Vertex Space, that reduces both the dimensionality and size of the required search space. Vertex Space ... mapping results in a reduced representation that serves as a characteristic target signature which is invariant to four of the six viewing geometry

49 CFR 213.369 - Inspection records.

Code of Federal Regulations, 2010 CFR

2010-10-01

... transfer records through electronic transmission, storage, and retrieval provided that— (1) The electronic... security such as recognition of an electronic signature, or other means, which uniquely identify the initiating person as the author of that record. No two persons shall have the same electronic identity; (2...

Developing Competitive Excellence in the Talented Female Athlete.

ERIC Educational Resources Information Center

Wildenhaus, Kevin J.

1995-01-01

This article reviews the historical development of female participation in sports, then identifies the unique issues associated with women's struggles for athletic acceptance and competitive excellence. Topics discussed include talent recognition and development, sex roles and socialization, physiology and maturation, coaching the female athlete,…

Mechanisms and neural basis of object and pattern recognition: a study with chess experts.

PubMed

Bilalić, Merim; Langner, Robert; Erb, Michael; Grodd, Wolfgang

2010-11-01

Comparing experts with novices offers unique insights into the functioning of cognition, based on the maximization of individual differences. Here we used this expertise approach to disentangle the mechanisms and neural basis behind two processes that contribute to everyday expertise: object and pattern recognition. We compared chess experts and novices performing chess-related and -unrelated (visual) search tasks. As expected, the superiority of experts was limited to the chess-specific task, as there were no differences in a control task that used the same chess stimuli but did not require chess-specific recognition. The analysis of eye movements showed that experts immediately and exclusively focused on the relevant aspects in the chess task, whereas novices also examined irrelevant aspects. With random chess positions, when pattern knowledge could not be used to guide perception, experts nevertheless maintained an advantage. Experts' superior domain-specific parafoveal vision, a consequence of their knowledge about individual domain-specific symbols, enabled improved object recognition. Functional magnetic resonance imaging corroborated this differentiation between object and pattern recognition and showed that chess-specific object recognition was accompanied by bilateral activation of the occipitotemporal junction, whereas chess-specific pattern recognition was related to bilateral activations in the middle part of the collateral sulci. Using the expertise approach together with carefully chosen controls and multiple dependent measures, we identified object and pattern recognition as two essential cognitive processes in expert visual cognition, which may also help to explain the mechanisms of everyday perception.

Sizing up the competition: quantifying the influence of the mental lexicon on auditory and visual spoken word recognition.

PubMed

Strand, Julia F; Sommers, Mitchell S

2011-09-01

Much research has explored how spoken word recognition is influenced by the architecture and dynamics of the mental lexicon (e.g., Luce and Pisoni, 1998; McClelland and Elman, 1986). A more recent question is whether the processes underlying word recognition are unique to the auditory domain, or whether visually perceived (lipread) speech may also be sensitive to the structure of the mental lexicon (Auer, 2002; Mattys, Bernstein, and Auer, 2002). The current research was designed to test the hypothesis that both aurally and visually perceived spoken words are isolated in the mental lexicon as a function of their modality-specific perceptual similarity to other words. Lexical competition (the extent to which perceptually similar words influence recognition of a stimulus word) was quantified using metrics that are well-established in the literature, as well as a statistical method for calculating perceptual confusability based on the phi-square statistic. Both auditory and visual spoken word recognition were influenced by modality-specific lexical competition as well as stimulus word frequency. These findings extend the scope of activation-competition models of spoken word recognition and reinforce the hypothesis (Auer, 2002; Mattys et al., 2002) that perceptual and cognitive properties underlying spoken word recognition are not specific to the auditory domain. In addition, the results support the use of the phi-square statistic as a better predictor of lexical competition than metrics currently used in models of spoken word recognition. © 2011 Acoustical Society of America

Self-identity reprogrammed by a single residue switch in a cell surface receptor of a social bacterium.

PubMed

Cao, Pengbo; Wall, Daniel

2017-04-04

The ability to recognize close kin confers survival benefits on single-celled microbes that live in complex and changing environments. Microbial kinship detection relies on perceptible cues that reflect relatedness between individuals, although the mechanisms underlying recognition in natural populations remain poorly understood. In myxobacteria, cells identify related individuals through a polymorphic cell surface receptor, TraA. Recognition of compatible receptors leads to outer membrane exchange among clonemates and fitness consequences. Here, we investigated how a single receptor creates a diversity in recognition across myxobacterial populations. We first show that TraA requires its partner protein TraB to function in cell-cell adhesion. Recognition is shown to be traA allele-specific, where polymorphisms within TraA dictate binding selectivity. We reveal the malleability of TraA recognition, and seemingly minor changes to its variable region reprogram recognition outcomes. Strikingly, we identify a single residue (A/P205) as a molecular switch for TraA recognition. Substitutions at this position change the specificity of a diverse panel of environmental TraA receptors. In addition, we engineered a receptor with unique specificity by simply creating an A205P substitution, suggesting that modest changes in TraA can lead to diversification of new recognition groups in nature. We hypothesize that the malleable property of TraA has allowed it to evolve and create social barriers between myxobacterial populations and in turn avoid adverse interactions with relatives.

Study on recognition algorithm for paper currency numbers based on neural network

NASA Astrophysics Data System (ADS)

Li, Xiuyan; Liu, Tiegen; Li, Yuanyao; Zhang, Zhongchuan; Deng, Shichao

2008-12-01

Based on the unique characteristic, the paper currency numbers can be put into record and the automatic identification equipment for paper currency numbers is supplied to currency circulation market in order to provide convenience for financial sectors to trace the fiduciary circulation socially and provide effective supervision on paper currency. Simultaneously it is favorable for identifying forged notes, blacklisting the forged notes numbers and solving the major social problems, such as armor cash carrier robbery, money laundering. For the purpose of recognizing the paper currency numbers, a recognition algorithm based on neural network is presented in the paper. Number lines in original paper currency images can be draw out through image processing, such as image de-noising, skew correction, segmentation, and image normalization. According to the different characteristics between digits and letters in serial number, two kinds of classifiers are designed. With the characteristics of associative memory, optimization-compute and rapid convergence, the Discrete Hopfield Neural Network (DHNN) is utilized to recognize the letters; with the characteristics of simple structure, quick learning and global optimum, the Radial-Basis Function Neural Network (RBFNN) is adopted to identify the digits. Then the final recognition results are obtained by combining the two kinds of recognition results in regular sequence. Through the simulation tests, it is confirmed by simulation results that the recognition algorithm of combination of two kinds of recognition methods has such advantages as high recognition rate and faster recognition simultaneously, which is worthy of broad application prospect.

Smart Materials Based on DNA Aptamers: Taking Aptasensing to the Next Level

PubMed Central

Mastronardi, Emily; Foster, Amanda; Zhang, Xueru; DeRosa, Maria C.

2014-01-01

“Smart” materials are an emerging category of multifunctional materials with physical or chemical properties that can be controllably altered in response to an external stimulus. By combining the standard properties of the advanced material with the unique ability to recognize and adapt in response to a change in their environment, these materials are finding applications in areas such as sensing and drug delivery. While the majority of these materials are responsive to physical or chemical changes, a particularly exciting area of research seeks to develop smart materials that are sensitive to specific molecular or biomolecular stimuli. These systems require the integration of a molecular recognition probe specific to the target molecule of interest. The ease of synthesis and labeling, low cost, and stability of DNA aptamers make them uniquely suited to effectively serve as molecular recognition probes in novel smart material systems. This review will highlight current work in the area of aptamer-based smart materials and prospects for their future applications. PMID:24553083

Ultra wide band 3-D cross section (RCS) holography

NASA Astrophysics Data System (ADS)

Collins, H. D.; Hall, T. E.

1992-07-01

Ultra wide band impulse holography is an exciting new concept for predictive radar cross section (RCS) evaluation employing near-field measurements. Reconstruction of the near-field hologram data maps the target's scattering areas, and uniquely identifies the 'hot spot' locations on the target. In addition, the target and calibration sphere's plane wave angular spectrums are computed (via digital algorithm) and used to generate the target's far-field RCS values in three dimensions for each frequency component in the impulse. Thin and thick targets are defined in terms of their near-field amplitude variations in range. Range gating and computer holographic techniques are applied to correct these variations. Preliminary experimental results on various targets verify the concept of RCS holography. The unique 3-D presentation (i.e., typically containing 524,288 RCS values for a 1024 (times) 512 sampled aperture for every frequency component) illustrates the efficacy of target recognition in terms of its far-field plane wave angular spectrum image. RCS images can then be viewed at different angles for target recognition, etc.

Tongue prints: A novel biometric and potential forensic tool.

PubMed

Radhika, T; Jeddy, Nadeem; Nithya, S

2016-01-01

Tongue is a vital internal organ well encased within the oral cavity and protected from the environment. It has unique features which differ from individual to individual and even between identical twins. The color, shape, and surface features are characteristic of every individual, and this serves as a tool for identification. Many modes of biometric systems have come into existence such as fingerprint, iris scan, skin color, signature verification, voice recognition, and face recognition. The search for a new personal identification method secure has led to the use of the lingual impression or the tongue print as a method of biometric authentication. Tongue characteristics exhibit sexual dimorphism thus aiding in the identification of the person. Emerging as a novel biometric tool, tongue prints also hold the promise of a potential forensic tool. This review highlights the uniqueness of tongue prints and its superiority over other biometric identification systems. The various methods of tongue print collection and the classification of tongue features are also elucidated.

Capturing specific abilities as a window into human individuality: The example of face recognition

PubMed Central

Wilmer, Jeremy B.; Germine, Laura; Chabris, Christopher F.; Chatterjee, Garga; Gerbasi, Margaret; Nakayama, Ken

2013-01-01

Proper characterization of each individual's unique pattern of strengths and weaknesses requires good measures of diverse abilities. Here, we advocate combining our growing understanding of neural and cognitive mechanisms with modern psychometric methods in a renewed effort to capture human individuality through a consideration of specific abilities. We articulate five criteria for the isolation and measurement of specific abilities, then apply these criteria to face recognition. We cleanly dissociate face recognition from more general visual and verbal recognition. This dissociation stretches across ability as well as disability, suggesting that specific developmental face recognition deficits are a special case of a broader specificity that spans the entire spectrum of human face recognition performance. Item-by-item results from 1,471 web-tested participants, included as supplementary information, fuel item analyses, validation, norming, and item response theory (IRT) analyses of our three tests: (a) the widely used Cambridge Face Memory Test (CFMT); (b) an Abstract Art Memory Test (AAMT), and (c) a Verbal Paired-Associates Memory Test (VPMT). The availability of this data set provides a solid foundation for interpreting future scores on these tests. We argue that the allied fields of experimental psychology, cognitive neuroscience, and vision science could fuel the discovery of additional specific abilities to add to face recognition, thereby providing new perspectives on human individuality. PMID:23428079

Unique associations between young adult men's emotional functioning and their body dissatisfaction and disordered eating.

PubMed

Griffiths, Scott; Angus, Douglas; Murray, Stuart B; Touyz, Stephen

2014-03-01

Research on emotional functioning, body dissatisfaction, and disordered eating in males is predominated by studies of negative affect and emotion regulation. Other aspects of emotional functioning, namely emotion recognition and attentional biases toward emotional stimuli, have received little empirical attention. The present study investigated the unique associations between different aspects of men's emotional functioning and their disordered eating attitudes, muscularity dissatisfaction, and body fat dissatisfaction. Results from 132 male undergraduates showed that muscularity dissatisfaction was uniquely associated with both emotion regulation difficulties and an attentional bias toward rejecting faces. Body fat dissatisfaction was not uniquely associated with any aspect of emotional functioning. Disordered eating was uniquely associated with emotion regulation difficulties. Collectively, the results indicate differences in the patterns of associations between men's emotional functioning and their body dissatisfaction and disordered eating. Copyright © 2013 Elsevier Ltd. All rights reserved.

Gold nanochestnut arrays as ultra-sensitive SERS substrate for detecting trace pesticide residue.

PubMed

Geng, Fei; Zhao, Huaping; Fu, Qun; Mi, Yan; Miao, Likun; Li, Wei; Dong, Yulian; Wu, Minghong; Lei, Yong

2018-07-20

In comparison to conventional spectroscopic techniques based on chromatography, surface-enhanced Raman spectroscopy (SERS) enables the rapid identification and detection of trace pesticide residues present in trace amounts in the environment and foods. Herein, a facile approach to fabricate unique gold nanochestnuts (GNCs) as an ultra-sensitive SERS substrate for detecting trace pesticide residues has been developed based on anodic aluminum oxide (AAO) templates. The GNCs are synthesized through the galvanic replacement of Ag on the top of Ni nanorod arrays. The as-prepared GNCs have well-controlled structural parameters, and importantly have unique anisotropic morphologies that benefit the enhancement in SERS performance. As a result, rhodamine 6 G (R6G) can be efficiently detected with GNCs as the SERS substrate even with a concentration of only 10 -12 M, and the Raman enhancement factor reaches up to 5.4 × 10 9 at this concentration. Further SERS measurement of thiram indicates a remarkable SERS-active sensitivity of the as-prepared GNCs with a detection limit of thiram up to 10 -14 M. The GNCs also exhibit a high signal-to-noise ratio.

Current opinion in Microbiology Roles of adaptor proteins in regulation of bacterial proteolysis

PubMed Central

Battesti, Aurelia; Gottesman, Susan

2013-01-01

Elimination of non-functional or unwanted proteins is critical for cell growth and regulation. In bacteria, ATP-dependent proteases target cytoplasmic proteins for degradation, contributing to both protein quality control and regulation of specific proteins, thus playing roles parallel to that of the proteasome in eukaryotic cells. Adaptor proteins provide a way to modulate the substrate specificity of the proteases and allow regulated proteolysis. Advances over the past few years have provided new insight into how adaptor proteins interact with both substrates and proteases and how adaptor functions are regulated. An important advance has come with the recognition of the critical roles of anti-adaptor proteins in regulating adaptor availability. PMID:23375660

The “parts and wholes” of face recognition: a review of the literature

PubMed Central

Tanaka, James W.; Simonyi, Diana

2016-01-01

It has been claimed that faces are recognized as a “whole” rather than the recognition of individual parts. In a paper published in the Quarterly Journal of Experimental Psychology in 1993, Martha Farah and I attempted to operationalize the holistic claim using the part/whole task. In this task, participants studied a face and then their memory presented in isolation and in the whole face. Consistent with the holistic view, recognition of the part was superior when tested in the whole-face condition compared to when it was tested in isolation. The “whole face” or holistic advantage was not found for faces that were inverted, or scrambled, nor for non-face objects suggesting that holistic encoding was specific to normal, intact faces. In this paper, we reflect on the part/whole paradigm and how it has contributed to our understanding of what it means to recognize a face as a “whole” stimulus. We describe the value of part/whole task for developing theories of holistic and non-holistic recognition of faces and objects. We discuss the research that has probed the neural substrates of holistic processing in healthy adults and people with prosopagnosia and autism. Finally, we examine how experience shapes holistic face recognition in children and recognition of own- and other-race faces in adults. The goal of this article is to summarize the research on the part/whole task and speculate on how it has informed our understanding of holistic face processing. PMID:26886495

The "parts and wholes" of face recognition: A review of the literature.

PubMed

Tanaka, James W; Simonyi, Diana

2016-10-01

It has been claimed that faces are recognized as a "whole" rather than by the recognition of individual parts. In a paper published in the Quarterly Journal of Experimental Psychology in 1993, Martha Farah and I attempted to operationalize the holistic claim using the part/whole task. In this task, participants studied a face and then their memory presented in isolation and in the whole face. Consistent with the holistic view, recognition of the part was superior when tested in the whole-face condition compared to when it was tested in isolation. The "whole face" or holistic advantage was not found for faces that were inverted, or scrambled, nor for non-face objects, suggesting that holistic encoding was specific to normal, intact faces. In this paper, we reflect on the part/whole paradigm and how it has contributed to our understanding of what it means to recognize a face as a "whole" stimulus. We describe the value of part/whole task for developing theories of holistic and non-holistic recognition of faces and objects. We discuss the research that has probed the neural substrates of holistic processing in healthy adults and people with prosopagnosia and autism. Finally, we examine how experience shapes holistic face recognition in children and recognition of own- and other-race faces in adults. The goal of this article is to summarize the research on the part/whole task and speculate on how it has informed our understanding of holistic face processing.

A Biologically Plausible Transform for Visual Recognition that is Invariant to Translation, Scale, and Rotation.

PubMed

Sountsov, Pavel; Santucci, David M; Lisman, John E

2011-01-01

Visual object recognition occurs easily despite differences in position, size, and rotation of the object, but the neural mechanisms responsible for this invariance are not known. We have found a set of transforms that achieve invariance in a neurally plausible way. We find that a transform based on local spatial frequency analysis of oriented segments and on logarithmic mapping, when applied twice in an iterative fashion, produces an output image that is unique to the object and that remains constant as the input image is shifted, scaled, or rotated.

A Biologically Plausible Transform for Visual Recognition that is Invariant to Translation, Scale, and Rotation

PubMed Central

Sountsov, Pavel; Santucci, David M.; Lisman, John E.

2011-01-01

Visual object recognition occurs easily despite differences in position, size, and rotation of the object, but the neural mechanisms responsible for this invariance are not known. We have found a set of transforms that achieve invariance in a neurally plausible way. We find that a transform based on local spatial frequency analysis of oriented segments and on logarithmic mapping, when applied twice in an iterative fashion, produces an output image that is unique to the object and that remains constant as the input image is shifted, scaled, or rotated. PMID:22125522

Picture object recognition in an American black bear (Ursus americanus).

PubMed

Johnson-Ulrich, Zoe; Vonk, Jennifer; Humbyrd, Mary; Crowley, Marilyn; Wojtkowski, Ela; Yates, Florence; Allard, Stephanie

2016-11-01

Many animals have been tested for conceptual discriminations using two-dimensional images as stimuli, and many of these species appear to transfer knowledge from 2D images to analogous real life objects. We tested an American black bear for picture-object recognition using a two alternative forced choice task. She was presented with four unique sets of objects and corresponding pictures. The bear showed generalization from both objects to pictures and pictures to objects; however, her transfer was superior when transferring from real objects to pictures, suggesting that bears can recognize visual features from real objects within photographic images during discriminations.

Emotional face recognition in adolescent suicide attempters and adolescents engaging in non-suicidal self-injury.

PubMed

Seymour, Karen E; Jones, Richard N; Cushman, Grace K; Galvan, Thania; Puzia, Megan E; Kim, Kerri L; Spirito, Anthony; Dickstein, Daniel P

2016-03-01

Little is known about the bio-behavioral mechanisms underlying and differentiating suicide attempts from non-suicidal self-injury (NSSI) in adolescents. Adolescents who attempt suicide or engage in NSSI often report significant interpersonal and social difficulties. Emotional face recognition ability is a fundamental skill required for successful social interactions, and deficits in this ability may provide insight into the unique brain-behavior interactions underlying suicide attempts versus NSSI in adolescents. Therefore, we examined emotional face recognition ability among three mutually exclusive groups: (1) inpatient adolescents who attempted suicide (SA, n = 30); (2) inpatient adolescents engaged in NSSI (NSSI, n = 30); and (3) typically developing controls (TDC, n = 30) without psychiatric illness. Participants included adolescents aged 13-17 years, matched on age, gender and full-scale IQ. Emotional face recognition was evaluated using the diagnostic assessment of nonverbal accuracy (DANVA-2). Compared to TDC youth, adolescents with NSSI made more errors on child fearful and adult sad face recognition while controlling for psychopathology and medication status (ps < 0.05). No differences were found on emotional face recognition between NSSI and SA groups. Secondary analyses showed that compared to inpatients without major depression, those with major depression made fewer errors on adult sad face recognition even when controlling for group status (p < 0.05). Further, compared to inpatients without generalized anxiety, those with generalized anxiety made fewer recognition errors on adult happy faces even when controlling for group status (p < 0.05). Adolescent inpatients engaged in NSSI showed greater deficits in emotional face recognition than TDC, but not inpatient adolescents who attempted suicide. Further results suggest the importance of psychopathology in emotional face recognition. Replication of these preliminary results and examination of the role of context-dependent emotional processing are needed moving forward.

2D DOST based local phase pattern for face recognition

NASA Astrophysics Data System (ADS)

Moniruzzaman, Md.; Alam, Mohammad S.

2017-05-01

A new two dimensional (2-D) Discrete Orthogonal Stcokwell Transform (DOST) based Local Phase Pattern (LPP) technique has been proposed for efficient face recognition. The proposed technique uses 2-D DOST as preliminary preprocessing and local phase pattern to form robust feature signature which can effectively accommodate various 3D facial distortions and illumination variations. The S-transform, is an extension of the ideas of the continuous wavelet transform (CWT), is also known for its local spectral phase properties in time-frequency representation (TFR). It provides a frequency dependent resolution of the time-frequency space and absolutely referenced local phase information while maintaining a direct relationship with the Fourier spectrum which is unique in TFR. After utilizing 2-D Stransform as the preprocessing and build local phase pattern from extracted phase information yield fast and efficient technique for face recognition. The proposed technique shows better correlation discrimination compared to alternate pattern recognition techniques such as wavelet or Gabor based face recognition. The performance of the proposed method has been tested using the Yale and extended Yale facial database under different environments such as illumination variation and 3D changes in facial expressions. Test results show that the proposed technique yields better performance compared to alternate time-frequency representation (TFR) based face recognition techniques.

A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

PubMed Central

2011-01-01

Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas. Results A metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

On the uniqueness of the receding contact angle: effects of substrate roughness and humidity on evaporation of water drops.

PubMed

Pittoni, Paola G; Lin, Chia-Hui; Yu, Teng-Shiang; Lin, Shi-Yow

2014-08-12

Could a unique receding contact angle be indicated for describing the wetting properties of a real gas-liquid-solid system? Could a receding contact angle be defined if the triple line of a sessile drop is not moving at all during the whole measurement process? To what extent is the receding contact angle influenced by the intrinsic properties of the system or the measurement procedures? In order to answer these questions, a systematic investigation was conducted in this study on the effects of substrate roughness and relative humidity on the behavior of pure water drops spreading and evaporating on polycarbonate (PC) surfaces characterized by different morphologies. Dynamic, advancing, and receding contact angles were found to be strongly affected by substrate roughness. Specifically, a receding contact angle could not be measured at all for drops evaporating on the more rugged PC surfaces, since the drops were observed strongly pinning to the substrate almost until their complete disappearance. Substrate roughness and system relative humidity were also found responsible for drastic changes in the depinning time (from ∼10 to ∼60 min). Thus, for measurement observations not sufficiently long, no movement of the triple line could be noted, with, again, the failure to find a receding contact angle. Therefore, to keep using concepts such as the receding contact angle as meaningful specifications of a given gas-liquid-solid system, the imperative to carefully investigate and report the inner characteristics of the system (substrate roughness, topography, impurities, defects, chemical properties, etc.) is pointed out in this study. The necessity of establishing methodological standards (drop size, measurement method, system history, observation interval, relative humidity, etc.) is also suggested.

Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, N.; Clay, M.D.; Belkum, M.J.van

2009-05-26

LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) andmore » N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the {alpha}-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C{sup {var_epsilon}} amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed by an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly, an apo-AtDAP-AT structure missing PLP revealed details of conformational changes induced by PLP binding and substrate entry into the active site.« less

Replica exchange molecular dynamics simulations provide insight into substrate recognition by small heat shock proteins.

PubMed

Patel, Sunita; Vierling, Elizabeth; Tama, Florence

2014-06-17

The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable α-helical character and contacts with the conserved α-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either "open" or "closed" based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone efficiency. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

PubMed Central

Wang, Wen-Jie; Cheng, Wang; Luo, Ming; Yan, Qingyu; Yu, Hong-Mei; Li, Qiong; Cao, Dong-Dong; Huang, Shengfeng; Xu, Anlong; Mariuzza, Roy A.; Chen, Yuxing; Zhou, Cong-Zhao

2015-01-01

Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum. PMID:26479246

Recollection and familiarity in amnesic mild cognitive impairment.

PubMed

Serra, Laura; Bozzali, Marco; Cercignani, Mara; Perri, Roberta; Fadda, Lucia; Caltagirone, Carlo; Carlesimo, Giovanni A

2010-05-01

To investigate whether, in patients with amnesic mild cognitive impairment (a-MCI), recognition deficits are mainly due to a selective impairment of recollection rather than familiarity. Nineteen patients with a-MCI and 23 sex-, age-, and education-matched healthy controls underwent two experimental investigations, using the Process Dissociation Procedure (PDP) and the Remember/Know (R/K) procedure, to assess the differential contribution of recollection and familiarity to their recognition performance. Both experimental procedures revealed a selective preservation of familiarity in a-MCI patients. Moreover, the R/K procedure showed a statistically significant impairment of recollection in a-MCI patients for words that were either read or anagrammed during the study phase. A-MCI is known to be commonly associated with a high risk of conversion to Alzheimer's disease (AD). Several previous studies have demonstrated a characteristic impairment of episodic memory in a-MCI, with an early dysfunction of recognition. Our findings are consistent with the knowledge of neurodegeneration occurring in AD, which is characterized, at the earliest disease stages, by a selective involvement of the entorhinal cortex. Moreover, the current study supports the dual process model of recognition, which hypothesizes recollection and familiarity to be independent processes associated with distinct anatomical substrates.

The medial prefrontal cortex-lateral entorhinal cortex circuit is essential for episodic-like memory and associative object-recognition.

PubMed

Chao, Owen Y; Huston, Joseph P; Li, Jay-Shake; Wang, An-Li; de Souza Silva, Maria A

2016-05-01

The prefrontal cortex directly projects to the lateral entorhinal cortex (LEC), an important substrate for engaging item-associated information and relaying the information to the hippocampus. Here we ask to what extent the communication between the prefrontal cortex and LEC is critically involved in the processing of episodic-like memory. We applied a disconnection procedure to test whether the interaction between the medial prefrontal cortex (mPFC) and LEC is essential for the expression of recognition memory. It was found that male rats that received unilateral NMDA lesions of the mPFC and LEC in the same hemisphere, exhibited intact episodic-like (what-where-when) and object-recognition memories. When these lesions were placed in the opposite hemispheres (disconnection), episodic-like and associative memories for object identity, location and context were impaired. However, the disconnection did not impair the components of episodic memory, namely memory for novel object (what), object place (where) and temporal order (when), per se. Thus, the present findings suggest that the mPFC and LEC are a critical part of a neural circuit that underlies episodic-like and associative object-recognition memory. © 2015 Wiley Periodicals, Inc.

Conserved conformational selection mechanism of Hsp70 chaperone-substrate interactions

PubMed Central

Velyvis, Algirdas; Zoltsman, Guy; Rosenzweig, Rina; Bouvignies, Guillaume

2018-01-01

Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event. PMID:29460778

Novel Substrate-Based Inhibitors of Human Glutamate Carboxypeptidase II with Enhanced Lipophilicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plechanovová, Anna; Byun, Youngjoo; Alquicer, Glenda

2012-10-09

Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance ofmore » nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1' specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.« less

Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

PubMed Central

Sakamoto, Yasumitsu; Suzuki, Yoshiyuki; Iizuka, Ippei; Tateoka, Chika; Roppongi, Saori; Fujimoto, Mayu; Inaka, Koji; Tanaka, Hiroaki; Yamada, Mitsugu; Ohta, Kazunori; Gouda, Hiroaki; Nonaka, Takamasa; Ogasawara, Wataru; Tanaka, Nobutada

2015-01-01

The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double β-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms. PMID:26057589

Phthalocyanine-BODIPY dye: synthesis, characterization, and utilization for pattern recognition of CYFRA 21-1 in whole blood samples.

PubMed

Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Yanık, Hülya; Göksel, Meltem; Alexandru, Anghel; Durmuş, Mahmut

2017-10-01

Phthalocyanine-BODIPY dye (BODIPY = boron dipyrromethene) was synthesized, fully characterized, and used for molecular recognition of CYFRA 21-1, a lung cancer biomarker, from whole blood samples. Thin films of three magnesium oxides ((MgO) n , where n = 8, 9, or 10)) were deposited on a paper substrate, and they were immersed in a solution of phthalocyanine-BODIPY dye (1.17 × 10 -3  mol/L) for the design of stochastic sensors. Limits of determination of picograms per milliliter magnitude order were recorded for the proposed stochastic sensors. CYFRA 21-1 was reliably identified and determined with recoveries higher than 95% and RSD lower than 1% in whole blood samples.

Impaired event memory and recollection in a case of developmental amnesia.

PubMed

Rosenbaum, R S; Carson, N; Abraham, N; Bowles, B; Kwan, D; Köhler, S; Svoboda, E; Levine, B; Richards, B

2011-10-01

A current debate in the literature is whether all declarative memories and associated memory processes rely on the same neural substrate. Here, we show that H.C., a developmental amnesic person with selective bilateral hippocampal volume loss, has a mild deficit in personal episodic memory, and a more pronounced deficit in public event memory; semantic memory for personal and general knowledge was unimpaired. This was accompanied by a subtle difference in impairment between recollection and familiarity on lab-based tests of recognition memory. Strikingly, H.C.'s recognition did not benefit from a levels-of-processing manipulation. Thus, not all types of declarative memory and related processes can exist independently of the hippocampus even if it is damaged early in life.

Testament of an Extraordinary Educator.

ERIC Educational Resources Information Center

Kulawiec, E. P.

1986-01-01

Examines the life and memoirs of Janusz Korczak (1878-1942), who attained considerable recognition as author, pediatrician, lecturer, publicist, philosopher, and educator. His greatest achievements were founding a unique, progressive child-raising method and directing an orphanage in the Warsaw Ghetto during World War II--until all were gassed by…

Recognition and Influence: The Evolution of Higher Education Research and Development

ERIC Educational Resources Information Center

Kandlbinder, Peter

2012-01-01

"Higher Education Research & Development" ("HERD") was established to address a perceived gap in higher education publishing: research of interest to practitioners that was engagingly written. After 30 years of contributing to the field, "HERD" has constructed a unique position for itself among higher education…

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease

PubMed Central

Robinson, Clifford R.; Sligar, Stephen G.

1998-01-01

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein–DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein–DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility. PMID:9482860

Constructed wetland using corncob charcoal substrate: pollutants removal and intensification.

PubMed

Liu, Mao; Li, Boyuan; Xue, Yingwen; Wang, Hongyu; Yang, Kai

2017-09-01

To investigate the feasibility of using corncob charcoal substrate in constructed wetlands, four laboratory-scale vertical flow constructed wetlands (VFCWs) were built. Effluent pollutant (chemical oxygen demand (COD), NH 4 + -N, total phosphorus (TP)) concentrations during the experiment were determined to reveal pollutant removal mechanisms and efficiencies at different stages. In the stable stage, a VFCW using clay ceramisite substrate under aeration attained higher COD (95.1%), and NH 4 + -N (95.1%) removal efficiencies than a VFCW using corncob charcoal substrate (91.5% COD, 91.3% NH 4 + -N) under aeration, but lower TP removal efficiency (clay ceramisite 32.0% and corncob charcoal 40.0%). The VFCW with raw corncob substrate showed stronger COD emissions (maximum concentration 3,108 mg/L) than the corncob charcoal substrate (COD was lower than influent). The VFCW using corncob charcoal substrate performed much better than the VFCW using clay ceramisite substrate under aeration when the C/N ratio was low (C/N = 1.5, TN removal efficiency 36.89%, 4.1% respectively). These results suggest that corncob charcoal is a potential substrate in VFCWs under aeration with a unique self -supplying carbon source property in the denitrification process.

Identification of Cyclin A Binders with a Fluorescent Peptide Sensor.

PubMed

Pazos, Elena; Mascareñas, José L; Vázquez, M Eugenio

2016-01-01

A peptide sensor that integrates the 4-dimethylaminophthalimide (4-DMAP) fluorophore in a short cyclin A binding sequence displays a large fluorescence emission increase upon interacting with the cyclin A Binding Groove (CBG). Competitive displacement assays of this probe allow the straightforward identification of peptides that interact with the CBG, which could potentially block the recognition of CDK/cyclin A kinase substrates.

The Neural Substrates of Recognition Memory for Verbal Information: Spanning the Divide between Short- and Long-Term Memory

ERIC Educational Resources Information Center

Buchsbaum, Bradley R.; Padmanabhan, Aarthi; Berman, Karen Faith

2011-01-01

One of the classic categorical divisions in the history of memory research is that between short-term and long-term memory. Indeed, because memory for the immediate past (a few seconds) and memory for the relatively more remote past (several seconds and beyond) are assumed to rely on distinct neural systems, more often than not, memory research…

Recognition of rotated images using the multi-valued neuron and rotation-invariant 2D Fourier descriptors

NASA Astrophysics Data System (ADS)

Aizenberg, Evgeni; Bigio, Irving J.; Rodriguez-Diaz, Eladio

2012-03-01

The Fourier descriptors paradigm is a well-established approach for affine-invariant characterization of shape contours. In the work presented here, we extend this method to images, and obtain a 2D Fourier representation that is invariant to image rotation. The proposed technique retains phase uniqueness, and therefore structural image information is not lost. Rotation-invariant phase coefficients were used to train a single multi-valued neuron (MVN) to recognize satellite and human face images rotated by a wide range of angles. Experiments yielded 100% and 96.43% classification rate for each data set, respectively. Recognition performance was additionally evaluated under effects of lossy JPEG compression and additive Gaussian noise. Preliminary results show that the derived rotation-invariant features combined with the MVN provide a promising scheme for efficient recognition of rotated images.

Pattern recognition of electronic bit-sequences using a semiconductor mode-locked laser and spatial light modulators

NASA Astrophysics Data System (ADS)

Bhooplapur, Sharad; Akbulut, Mehmetkan; Quinlan, Franklyn; Delfyett, Peter J.

2010-04-01

A novel scheme for recognition of electronic bit-sequences is demonstrated. Two electronic bit-sequences that are to be compared are each mapped to a unique code from a set of Walsh-Hadamard codes. The codes are then encoded in parallel on the spectral phase of the frequency comb lines from a frequency-stabilized mode-locked semiconductor laser. Phase encoding is achieved by using two independent spatial light modulators based on liquid crystal arrays. Encoded pulses are compared using interferometric pulse detection and differential balanced photodetection. Orthogonal codes eight bits long are compared, and matched codes are successfully distinguished from mismatched codes with very low error rates, of around 10-18. This technique has potential for high-speed, high accuracy recognition of bit-sequences, with applications in keyword searches and internet protocol packet routing.

Bursting at the Seams: Rippled Monolayer Bismuth on NbSe 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Alan; Adamo, Carolina; Jia, Shuang

Bismuth, one of the heaviest semimetals in nature, ignited the interest of the materials-physics community for its potential impact on topological quantum-material systems that utilize its strong spin-orbit coupling (SOC) and unique orbital hybridization. In particular, recent theoretical predictions of unique topological and superconducting properties of thin bismuth films and interfaces prompted intense research on the growth of sub- to a few monolayers of bismuth on different substrates. Similar to bulk rhombohedral bismuth, the initial growth of bismuth films on most substrates results in buckled bilayers that either grow in the (111) or (110) directions, with a lattice constant closemore » to that of bulk Bi. By contrast, in this paper we show a new growth pattern for bismuth monolayers on NbSe 2. We find that the initial growth of Bi can form a strongly bonded commensurate layer, resulting in a compressively strained two-dimensional triangular lattice. A unique pattern of 1D ripples and domain walls is observed. The single layer of bismuth also introduces strong marks on the electronic properties at the surface.« less

Bursting at the Seams: Rippled Monolayer Bismuth on NbSe 2

DOE PAGES

Fang, Alan; Adamo, Carolina; Jia, Shuang; ...

2018-04-13

Bismuth, one of the heaviest semimetals in nature, ignited the interest of the materials-physics community for its potential impact on topological quantum-material systems that utilize its strong spin-orbit coupling (SOC) and unique orbital hybridization. In particular, recent theoretical predictions of unique topological and superconducting properties of thin bismuth films and interfaces prompted intense research on the growth of sub- to a few monolayers of bismuth on different substrates. Similar to bulk rhombohedral bismuth, the initial growth of bismuth films on most substrates results in buckled bilayers that either grow in the (111) or (110) directions, with a lattice constant closemore » to that of bulk Bi. By contrast, in this paper we show a new growth pattern for bismuth monolayers on NbSe 2. We find that the initial growth of Bi can form a strongly bonded commensurate layer, resulting in a compressively strained two-dimensional triangular lattice. A unique pattern of 1D ripples and domain walls is observed. The single layer of bismuth also introduces strong marks on the electronic properties at the surface.« less

Robust Real-Time and Rotation-Invariant American Sign Language Alphabet Recognition Using Range Camera

NASA Astrophysics Data System (ADS)

Lahamy, H.; Lichti, D.

2012-07-01

The automatic interpretation of human gestures can be used for a natural interaction with computers without the use of mechanical devices such as keyboards and mice. The recognition of hand postures have been studied for many years. However, most of the literature in this area has considered 2D images which cannot provide a full description of the hand gestures. In addition, a rotation-invariant identification remains an unsolved problem even with the use of 2D images. The objective of the current study is to design a rotation-invariant recognition process while using a 3D signature for classifying hand postures. An heuristic and voxelbased signature has been designed and implemented. The tracking of the hand motion is achieved with the Kalman filter. A unique training image per posture is used in the supervised classification. The designed recognition process and the tracking procedure have been successfully evaluated. This study has demonstrated the efficiency of the proposed rotation invariant 3D hand posture signature which leads to 98.24% recognition rate after testing 12723 samples of 12 gestures taken from the alphabet of the American Sign Language.