highly-sensitive-rapid-fluorescence-detection-with-portable-fret

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

Konyang University

High-throughput FACS method for directed evolution of substrate specificity.

&#x7CBE;&#x81F4;&#x7684;&#x9009;&#x62E9;&#x6027;&#x548C;&#x50AC;&#x5316;&#x6D3B;&#x6027;&#x7684;&#x9176;&#x5DF2;&#x7ECF;&#x5F62;&#x6210;&#x7684;&#x79EF;&#x6781;&#x548C;&#x6D88;&#x6781;&#x7684;&#x9009;&#x62E9;&#x538B;&#x529B;&#x5F71;&#x54CD;&#x7684;&#x6F14;&#x53D8;&#x8FC7;&#x7A0B;&#x4E2D;&#x3002;&#x76F8;&#x6BD4;&#x4E4B;&#x4E0B;&#xFF0C;&#x5DE5;&#x7A0B;&#x4F7F;&#x7528;&#x4F53;&#x5916;&#x7B5B;&#x9009;&#x6280;&#x672F;&#x901A;&#x5E38;&#x63A5;&#x53D7;&#x65B0;&#x578B;&#x57FA;&#x8D28;&#x9176;&#x53D8;&#x4F53;&#x663E;&#x793A;&#x8F83;&#x9AD8;&#x7A0B;&#x5EA6;&#x7684;&#x50AC;&#x5316;&#x6EE5;&#x4EA4;&#x4E0E;&#x81EA;&#x7136;&#x540C;&#x884C;&#x8F83;&#x4F4E;&#x7684;&#x603B;&#x8425;&#x4E1A;&#x989D;&#x3002;&#x4F7F;&#x7528;&#x7EC6;&#x83CC;&#x663E;&#x793A;&#x548C;&#x591A;&#x53C2;&#x6570;&#x6D41;&#x5F0F;&#x7EC6;&#x80DE;&#x672F;&#xFF0C;&#x6211;&#x4EEC;&#x5F00;&#x53D1;&#x4E86;&#x6A21;&#x62DF;&#x4F53;&#x5916;&#x7684;&#x9176;&#x53D8;&#x4F53;&#x5206;&#x79BB;&#x7684;&#x79EF;&#x6781;&#x548C;&#x6D88;&#x6781;&#x9009;&#x62E9;&#x6027;&#x538B;&#x529B;&#x4E0E;&#x53CD;&#x5E94;&#x6027;&#x6240;&#x9700;&#x7684;&#x65B0;&#x578B;&#x886C;&#x5E95;&#xFF0C;&#x800C;&#x540C;&#x65F6;&#x6392;&#x9664;&#x90A3;&#x4E9B;&#x4E0E;&#x53CD;&#x5E94;&#x6027;&#x610F;&#x5916;&#x886C;&#x5E95;&#x671D;&#x7684;&#x65B0;&#x9896;&#x65B9;&#x6CD5;&#x3002;&#x5927;&#x578B;&#x7684;&#x5927;&#x80A0;&#x57C3;&#x5E0C;&#x6C0F;&#x5927;&#x80A0;&#x6746;&#x83CC;&#x5185;&#x80BD;&#x9176; OmpT &#x7684;&#x968F;&#x673A;&#x7A81;&#x53D8;&#x4F53;&#x5E93;&#x7684;&#x7B5B;&#x9009;&#x5BFC;&#x81F4;&#x7684;&#x9176; variant&#xFF0C;1.3.19&#xFF0C;&#x5206;&#x89E3; Ala Arg &#x80BD;&#x952E;&#x800C;&#x4E0D;&#x662F;&#x9996;&#x9009;&#x7684; WT &#x9176;&#x7684; Arg Arg &#x503A;&#x5238;&#x5206;&#x79BB;&#x3002;&#x53D8;&#x578B; 1.3.19 &#x5C55;&#x51FA;&#x5927;&#x4E8E;&#x4E09;&#x4E2A;&#x7FFB;&#x5929;&#x8986;&#x5730;&#x7684;&#x9009;&#x62E9;&#x6027; &#xFF08;-Ala-Arg-/-&#x7CBE;&#x6C28;&#x9178;-&#x7CBE;&#x6C28;&#x9178;-) &#x548C;&#x4E3A;&#x6240;&#x663E;&#x793A;&#x7684;&#x9996;&#x9009;&#x886C;&#x5E95;&#xFF0C;Arg &#x53C2;&#x6570;&#x7684;&#x7236;&#x7EA7;&#x76F8;&#x540C;&#x7684; Ala Arg &#x5288;&#x88C2;&#x7684;&#x50AC;&#x5316;&#x6548;&#x7387;Ser223Arg &#x66FF;&#x6362;&#x5355;&#x4E2A;&#x6C28;&#x57FA;&#x9178;&#x6240;&#x793A;&#x5B8C;&#x5168;&#x8BB2;&#x8FF0;&#x72EC;&#x7279;&#x7684;&#x50AC;&#x5316;&#x6027;&#x80FD;&#xFF0C;1.3.19 &#x7684; variant &#x7C7B;&#x578B;&#x7684;&#x503C;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x53EF;&#x4EE5;&#x89E3;&#x91CA;&#x63D0;&#x51FA;&#x8FD9;&#x4E2A;&#x7A81;&#x53D8;&#x7684;&#x884C;&#x4E3A;&#x662F;&#x4EE5;&quot;&#x4EA4;&#x6362;&quot;&#x901A;&#x5E38; WT &#x57FA;&#x677F;&#x80BD;&#x4E0E; 223Arg &#x4FA7;&#x94FE;&#x5728;&#x53D1;&#x73B0; OmpT S(1) &#x5B50;&#x7F51;&#x7AD9; P(1) Arg &#x4FA7;&#x94FE;&#x3002;

&#x5DE5;&#x7A0B;&#x5C55;&#x793A;&#x9AD8;&#x7684;&#x50AC;&#x5316;&#x6D3B;&#x6027;&#x548C;&#x7CBE;&#x81F4;&#x7684;&#x57FA;&#x677F;&#x9009;&#x62E9;&#x6027;&#x7684;&#x86CB;&#x767D;&#x9176;&#x53D8;&#x79CD;&#x3002;

The exquisite selectivity and catalytic activity of enzymes have been shaped by the effects of positive and negative selection pressure during the course of evolution. In contrast, enzyme variants engineered by using in vitro screening techniques to accept novel substrates typically display a higher degree of catalytic promiscuity and lower total turnover in comparison with their natural counterparts. Using bacterial display and multiparameter flow cytometry, we have developed a novel methodology for emulating positive and negative selective pressure in vitro for the isolation of enzyme variants with reactivity for desired novel substrates, while simultaneously excluding those with reactivity toward undesired substrates. Screening of a large library of random mutants of the Escherichia coli endopeptidase OmpT led to the isolation of an enzyme variant, 1.3.19, that cleaved an Ala-Arg peptide bond instead of the Arg-Arg bond preferred by the WT enzyme. Variant 1.3.19 exhibited greater than three million-fold selectivity (-Ala-Arg-/-Arg-Arg-) and a catalytic efficiency for Ala-Arg cleavage that is the same as that displayed by the parent for the preferred substrate, Arg-Arg. A single amino acid Ser223Arg substitution was shown to recapitulate completely the unique catalytic properties of the 1.3.19 variant. These results can be explained by proposing that this mutation acts to "swap" the P(1) Arg side chain normally found in WT substrate peptides with the 223Arg side chain in the S(1) subsite of OmpT.

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity.

La s&eacute;lectivit&eacute; exquise et l&#39;activit&eacute; catalytique des enzymes ont &eacute;t&eacute; fa&ccedil;onn&eacute;s par les effets de la pression de s&eacute;lection positive et n&eacute;gative au cours de l&#39;&eacute;volution. En revanche, variantes d&#39;enzyme con&ccedil;us en utilisant des techniques de d&eacute;pistage in vitro d&#39;accepter de nouveaux substrats g&eacute;n&eacute;ralement affichent une plus grande promiscuit&eacute; catalytique et chiffre d&#39;affaires inf&eacute;rieur en comparaison avec leurs homologues naturels. Utilisez affichage bact&eacute;rienne et cytom&eacute;trie en flux multiparam&eacute;trique, nous avons d&eacute;velopp&eacute; une m&eacute;thodologie novatrice pour &eacute;muler positif et n&eacute;gatif de pression de s&eacute;lection in vitro pour l&#39;isolement des variantes de l&#39;enzyme avec r&eacute;activit&eacute; pour d&eacute;sir&eacute; nouveaux substrats, tandis que simultan&eacute;ment &agrave; l&#39;exclusion de ceux avec r&eacute;activit&eacute; vis-&agrave;-vis des substrats non d&eacute;sir&eacute;s. D&eacute;pistage d&#39;une grande biblioth&egrave;que de mutants al&eacute;atoires de l&#39;endopeptidase de coli Escherichia od&egrave;le conduit &agrave; l&#39;isolement d&#39;une variante de l&#39;enzyme, 1.3.19, qui s&#39;est fendue d&#39;une liaison peptidique de Ala-Arg au lieu de la liaison Arg-Arg pr&eacute;f&eacute;r&eacute;e par l&#39;enzyme WT. 1.3.19 Variante expos&eacute; sup&eacute;rieur &agrave; trois amplifiant s&eacute;lectivit&eacute; (- Ala - Arg-/ - Arg - Arg-) et une efficacit&eacute; catalytique pour le clivage Ala-Arg c&#39;est la m&ecirc;me que celle affich&eacute;e par le parent pour le substrat pr&eacute;f&eacute;r&eacute;, Arg-arg. Un seul acide amin&eacute; Ser223Arg substitution s&#39;est av&eacute;r&eacute; r&eacute;capituler compl&egrave;tement uniques propri&eacute;t&eacute;s catalytiques de la 1.3.19 variante. Ces r&eacute;sultats peuvent &ecirc;tre expliqu&eacute;s en proposant que cette mutation agit pour la cha&icirc;ne lat&eacute;rale Arg p se trouv&eacute;e normalement dans les peptides substrat WT avec la cha&icirc;ne lat&eacute;rale de 223Arg dans le sous-site s (1) d&#39;od&egrave;le de &laquo; swap &raquo;.

Ing&eacute;nierie des variantes de prot&eacute;ase pr&eacute;sentant une forte activit&eacute; catalytique et s&eacute;lectivit&eacute; de substrat exquis.

Die exquisite Selektivit&auml;t und katalytische Aktivit&auml;t der Enzyme haben im Laufe der Evolution durch die Auswirkungen der positiven und negativen Selektionsdruck gepr&auml;gt. Im Gegensatz dazu anzeigen Enzymvarianten entwickelt, die mithilfe von in-vitro-Screening-Techniken neuartige Substrate akzeptieren in der Regel ein h&ouml;heres Ma&szlig; an katalytische Promiskuit&auml;t und niedrigeren Gesamtumsatz im Vergleich zu ihren nat&uuml;rlichen Gegenst&uuml;cken. Mit bakteriellen Display und multiparameter Durchflusszytometrie, entwickelten wir eine neuartige Methode zur Emulation von positiven und negativen selektiven Druck in-vitro f&uuml;r die Isolierung von Enzymvarianten mit Reaktivit&auml;t f&uuml;r gew&uuml;nschte Roman Substrate, w&auml;hrend gleichzeitig mit Reaktivit&auml;t gegen unerw&uuml;nschte Untergr&uuml;nde ausgenommen. Screening von einer umfangreichen Bibliothek von zuf&auml;lligen Mutanten von Escherichia coli Endopeptidase OmpT f&uuml;hrte zur Isolierung eine Enzym-Variante 1.3.19, die eine Ala-Arg-Peptidbindung anstelle der Arg-Arg-Bond bevorzugt durch die WT-Enzym zerspaltet. Variant 1.3.19 ausgestellt gr&ouml;&szlig;er als drei millionenfache Selektivit&auml;t (- Ala - Arg-/ - Arg - Arg-) und eine katalytische Effizienz f&uuml;r Ala-Arg-Spaltung, die identisch angezeigt, indem das &uuml;bergeordnete Element f&uuml;r das bevorzugte Substrat, Arg-arg. Eine einzige Aminos&auml;ure Ser223Arg Ersetzung wurde gezeigt, dass die einzigartigen katalytischen Eigenschaften von der 1.3.19 vollst&auml;ndig rekapitulieren Variante. Diese Ergebnisse k&ouml;nnen erkl&auml;rt werden, indem er vorschl&auml;gt, dass diese Mutation handelt, um &quot;die Seitenkette P(1) Arg normalerweise gefunden in WT Substrat Peptide mit der 223Arg-Seitenkette in der S(1)-Subsite von OmpT tauschen&quot;.

Engineering von Protease-Varianten, die hohe katalytische Aktivit&auml;t und exquisite Substrat Selektivit&auml;t ausstellen.

La selettivit&agrave; squisito e attivit&agrave; catalitica degli enzimi sono state modellate dagli effetti della pressione di selezione positiva e negativa nel corso dell&#39;evoluzione. Al contrario, varianti di enzima progettati utilizzando tecniche di screening in vitro ad per accettare nuovi substrati in genere visualizzano un maggiore grado di promiscuit&agrave; catalitico e fatturato totale inferiore rispetto alle loro controparti naturali. Mediante batterica e la citometria a flusso multiparametrico, abbiamo sviluppato una metodologia romanzo per emulare la pressione selettiva positiva e negativa in vitro per l&#39;isolamento delle varianti di enzima con reattivit&agrave; per substrati romanzo desiderati, mentre contemporaneamente escludendo quelli con reattivit&agrave; verso substrati indesiderati. Lo screening di una vasta libreria di mutanti casuali dell&#39;endopeptidasi coli Escherichia salvataggi ha condotto all&#39;isolamento di una variante di enzima, 1.3.19, che scisse un legame peptidico Ala-Arg invece il legame Arg-Arg preferito dall&#39;enzima WT. 1.3.19 Variante esposta maggiore di tre milioni di selettivit&agrave; (- Ala - Arg-/ - Arg - Arg-) e un&#39;efficienza catalitica per Ala-Arg scissione che &egrave; uguale a quello visualizzato dal padre per il substrato preferito, Arg-arg. Un singolo amminoacido Ser223Arg sostituzione &egrave; stato indicato per ripassare completamente l&#39;unica propriet&agrave; catalitiche del 1.3.19 variante. Questi risultati possono essere spiegati da proporre che questa mutazione agisce per &quot;scambiare&quot; la catena laterale P(1) Arg normalmente si trovano in peptidi substrato WT con catena laterale 223Arg nel S(1) sito secondario di salvataggi.

Ingegneria delle varianti di proteasi espositrici ad alta attivit&agrave; catalitica e selettivit&agrave; del substrato squisito.

&#x7D76;&#x5999;&#x306A;&#x9078;&#x629E;&#x6027;&#x3068;&#x89E6;&#x5A92;&#x306E;&#x6D3B;&#x6027;&#x9175;&#x7D20;&#x306E;&#x6B63;&#x3068;&#x8CA0;&#x306E;&#x9078;&#x629E;&#x5727;&#x306E;&#x5F71;&#x97FF;&#x306B;&#x3088;&#x3063;&#x3066;&#x9032;&#x5316;&#x306E;&#x904E;&#x7A0B;&#x3067;&#x5F62;&#x3092;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5BFE;&#x7167;&#x7684;&#x306B;&#x3001;&#x9175;&#x7D20;&#x306E;&#x4E9C;&#x7A2E;&#x306E; in vitro &#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x6280;&#x8853;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x901A;&#x5E38;&#x65B0;&#x3057;&#x3044;&#x57FA;&#x677F;&#x3092;&#x53D7;&#x3051;&#x5165;&#x308C;&#x308B;&#x3088;&#x3046;&#x306B;&#x8A2D;&#x8A08;&#x3055;&#x308C;&#x3066;&#x3088;&#x308A;&#x9AD8;&#x5EA6;&#x306A;&#x89E6;&#x5A92;&#x4E71;&#x4EA4;&#x3068;&#x305D;&#x306E;&#x81EA;&#x7136;&#x306E;&#x56FD;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x4F4E;&#x3044;&#x7DCF;&#x58F2;&#x308A;&#x4E0A;&#x3052;&#x9AD8;&#x3092;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D30;&#x83CC;&#x306E;&#x30C7;&#x30A3;&#x30B9;&#x30D7;&#x30EC;&#x30A4;&#x3068;&#xFF0C; &#x30D5;&#x30ED;&#x30FC;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30C8;&#x30EA;&#x30FC;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x6B63;&#x3068;&#x8CA0;&#x306E;&#x9078;&#x629E;&#x5727;&#x4F53;&#x5916;&#x9175;&#x7D20;&#x306E;&#x4E9C;&#x7A2E;&#x306E;&#x7D76;&#x7E01;&#x76EE;&#x7684;&#x5C0F;&#x8AAC;&#x7528;&#x53CD;&#x5FDC;&#x6027;&#x3068;&#x540C;&#x6642;&#x306B;&#x671B;&#x307E;&#x3057;&#x304F;&#x306A;&#x3044;&#x57FA;&#x677F;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x53CD;&#x5FDC;&#x6027;&#x3068;&#x306E;&#x305D;&#x308C;&#x3089;&#x3092;&#x6392;&#x9664;&#x3057;&#x3066;&#x3001;&#x30A8;&#x30DF;&#x30E5;&#x30EC;&#x30FC;&#x30C8;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x65B0;&#x3057;&#x3044;&#x65B9;&#x6CD5;&#x8AD6;&#x3092;&#x958B;&#x767A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x5927;&#x8178;&#x83CC;&#x5927;&#x8178;&#x83CC;&#x30A8;&#x30F3;&#x30C9;&#x30DA;&#x30D7;&#x30C1;&#x30C0;&#x30FC;&#x30BC; OmpT &#x306E;&#x30E9;&#x30F3;&#x30C0;&#x30E0;&#x306A;&#x7A81;&#x7136;&#x5909;&#x7570;&#x4F53;&#x306E;&#x5927;&#x898F;&#x6A21;&#x306A;&#x30E9;&#x30A4;&#x30D6;&#x30E9;&#x30EA;&#x306E;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x306F;&#x3001;Ala Arg &#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x7D50;&#x5408; WT &#x9175;&#x7D20;&#x306B;&#x3088;&#x3063;&#x3066;&#x512A;&#x5148;&#x5F15;&#x6570; Arg &#x50B5;&#x5238;&#x306E;&#x4EE3;&#x308F;&#x308A;&#x306B;&#x5207;&#x65AD;&#x3001;&#x9175;&#x7D20; variant&#x3001;1.3.19 &#x306E;&#x5206;&#x96E2;&#x306B;&#x3064;&#x306A;&#x304C;&#x3063;&#x305F;&#x3002;&#x30D0;&#x30EA;&#x30A2;&#x30F3;&#x30C8; 1.3.19 &#x5C55;&#x793A;&#x4EE5;&#x4E0A;&#x767A;&#x660E;&#x8005;&#x9078;&#x629E;&#x6027; (-Ala Arg-/- &#x30A2;&#x30EB;&#x30BC;&#x30F3;&#x30C1;&#x30F3; - &#x30A2;&#x30EB;&#x30BC;&#x30F3;&#x30C1;&#x30F3;-) &#x3068;&#x306F;&#x3001;&#x6700;&#x5BC4;&#x308A;&#x306E;&#x57FA;&#x677F;&#x306F;&#x3001;Arg &#x5F15;&#x6570;&#x89AA;&#x306B;&#x8868;&#x793A;&#x3068;&#x540C;&#x3058;&#x7FFC; Arg &#x80F8;&#x306E;&#x8C37;&#x9593;&#x306E;&#x89E6;&#x5A92;&#x4F5C;&#x7528;&#x306E;&#x52B9;&#x7387;&#x306F;&#x3001;&#x3002;&#x5358;&#x4E00;&#x306E;&#x30A2;&#x30DF;&#x30CE;&#x9178; Ser223Arg &#x7F6E;&#x63DB;&#x3001;1.3.19 &#x306E;&#x30E6;&#x30CB;&#x30FC;&#x30AF;&#x306A;&#x89E6;&#x5A92;&#x4F5C;&#x7528;&#x3092;&#x5B8C;&#x5168;&#x306B;&#x8981;&#x7D04;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x3055;&#x308C;&#x305F;&#x30D0;&#x30EA;&#x30A2;&#x30F3;&#x30C8;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x3053;&#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&quot;&#x901A;&#x5E38; WT &#x57FA;&#x8CEA;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x306E; 223Arg &#x5074;&#x9396;&#x306B; OmpT &#x306E; S(1) &#x30B5;&#x30D6;&#x30B5;&#x30A4;&#x30C8;&#x3067;&#x767A;&#x898B; P(1) &#x30A2;&#x30EB;&#x30BC;&#x30F3;&#x30C1;&#x30F3;&#x5074;&#x9396;&#x3092;&#x30B9;&#x30EF;&#x30C3;&#x30D7;&#x3059;&#x308B;&#x306B;&#x306F;&quot;&#x884C;&#x52D5;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x63D0;&#x6848;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x8AAC;&#x660E;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;

&#x9AD8;&#x3044;&#x89E6;&#x5A92;&#x6D3B;&#x6027;&#x3068;&#x7D76;&#x5999;&#x306A;&#x57FA;&#x8CEA;&#x9078;&#x629E;&#x6027;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x306E;&#x4E9C;&#x7A2E;&#x306E;&#x30A8;&#x30F3;&#x30B8;&#x30CB;&#x30A2; &#x30EA;&#x30F3;&#x30B0;&#x3002;

&#xC808;&#xBB18; &#xD55C; &#xC120;&#xD0DD;&#xB3C4; &#xBC0F; &#xD6A8;&#xC18C;&#xC758; &#xCD09;&#xB9E4; &#xD65C;&#xB3D9; &#xC9C4;&#xD654; &#xACFC;&#xC815;&#xC758; &#xAE0D;&#xC815; &#xBC0F; &#xBD80;&#xC815; &#xC120;&#xD0DD; &#xC555;&#xB825; &#xD6A8;&#xACFC; &#xC758;&#xD574; &#xD615;&#xC131; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xBC18;&#xBA74;, &#xD6A8;&#xC18C; &#xBCC0;&#xC885; &#xC18C;&#xC124; &#xAE30;&#xD310;&#xC5D0; &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; &#xB3D9;&#xC758; &#xCCB4; &#xC678; &#xAC80;&#xC0AC; &#xAE30;&#xBC95;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC124;&#xACC4; &#xB41C; &#xCD09;&#xB9E4; &#xC131;&#xD589;&#xC704;&#xC640; &#xC790;&#xC5F0; &#xB4E4;&#xC5D0; &#xBE44;&#xD574; &#xB0AE;&#xC740; &#xCD1D; &#xB9E4;&#xCD9C;&#xC758; &#xB192;&#xC740; &#xD559;&#xC704;&#xB97C; &#xD45C;&#xC2DC; &#xD569;&#xB2C8;&#xB2E4;. Multiparameter cytometry &#xC138;&#xADE0; &#xB514;&#xC2A4;&#xD50C;&#xB808;&#xC774; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xB3D9;&#xC2DC;&#xC5D0; &#xC6D0;&#xCE58; &#xC54A;&#xB294; &#xAE30;&#xD310; &#xCABD;&#xC73C;&#xB85C; &#xBC18;&#xC751;&#xC5D0; &#xADF8;&#xB4E4;&#xC744; &#xC81C;&#xC678; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xC6D0;&#xD558;&#xB294; &#xC18C;&#xC124; &#xAE30;&#xD310;&#xC5D0; &#xB300; &#xD55C; &#xBC18;&#xC751;&#xC73C;&#xB85C; &#xAE0D;&#xC815;&#xC801;&#xC774; &#xACE0; &#xBD80;&#xC815;&#xC801;&#xC778; &#xC120;&#xD0DD;&#xC801; &#xC555;&#xB825; &#xD6A8;&#xC18C; &#xBCC0;&#xC885;&#xC758; &#xACA9;&#xB9AC;&#xC5D0; &#xB300; &#xD55C; &#xCCB4; &#xC678;&#xB97C; &#xBAA8;&#xBC29; &#xD558;&#xB294; &#xAC83;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xBC29;&#xBC95;&#xB860;&#xC744; &#xAC1C;&#xBC1C; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xBCD1;&#xC6D0; &#xC131; &#xB300;&#xC7A5;&#xADE0; endopeptidase Ompt&#xC758; &#xBB34;&#xC791;&#xC704; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; &#xD070; &#xB3C4;&#xC11C;&#xAD00;&#xC758; &#xC2EC;&#xC0AC;&#xB294; &#xD6A8;&#xC18C; &#xBCC0;&#xC885;, 1.3.19, WT &#xD6A8;&#xC18C;&#xC5D0; &#xC758;&#xD574; &#xC120;&#xD638; Arg Arg &#xBCF8;&#xB4DC; &#xB300;&#xC2E0; Ala Arg &#xD3A9; &#xD2F0; &#xB4DC; &#xACB0;&#xD569;&#xC744; &#xD2B9;&#xD788; &#xACA9;&#xB9AC;&#xB97C; &#xC774;&#xB04C;&#xC5C8;&#xB2E4;. &#xBCC0;&#xD615; 1.3.19 &#xC804;&#xC2DC; 3 &#xBCF4;&#xB2E4; &#xD070; million-fold &#xC120;&#xD0DD;&#xB3C4; (-&#xD06C; &#xB78C;-Arg-/-Arg-Arg-)&#xC640; &#xAE30;&#xBCF8; &#xAE30;&#xD310; Arg-arg.&#xC5D0; &#xB300; &#xD55C; &#xBD80;&#xBAA8;&#xC5D0; &#xC758;&#xD574; &#xD45C;&#xC2DC; &#xB418;&#xB294; &#xACB0;&#xACFC;&#xC640; Ala Arg &#xBD84;&#xC5F4;&#xC5D0; &#xB300; &#xD55C; &#xCD09;&#xB9E4; &#xD6A8;&#xC728; &#xB2E8;&#xC77C; &#xC544;&#xBBF8;&#xB178;&#xC0B0; Ser223Arg &#xB300;&#xCCB4; &#xD569;&#xB2C8;&#xB2E4; 1.3.19&#xC758; &#xB3C5;&#xD2B9;&#xD55C; &#xCD09;&#xB9E4; &#xD2B9;&#xC131;&#xC744; &#xC644;&#xC804;&#xD788; &#xC815;&#xB9AC; &#xC92C;&#xB358; &#xBCC0;&#xC885;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC;&#xC774; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &quot;&#xAD50;&#xD658;&quot; &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; WT &#xAE30;&#xD310; &#xD3A9; &#xD2F0; &#xB4DC;&#xC5D0;&#xC11C; 223Arg &#xC0AC;&#xC774;&#xB4DC; &#xCCB4;&#xC778; Ompt&#xC758; S(1) &#xD558;&#xC704; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0;&#xC11C; &#xBC1C;&#xACAC; P(1) Arg &#xC0AC;&#xC774;&#xB4DC; &#xCCB4;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xC81C;&#xC548; &#xD558; &#xC5EC; &#xC124;&#xBA85;&#xD560; &#xC218; &#xC788;&#xB2E4;.

&#xB192;&#xC740; &#xCD09;&#xB9E4; &#xD65C;&#xB3D9; &#xBC0F; &#xC808;&#xBB18; &#xD55C; &#xAE30;&#xD310; &#xC120;&#xD0DD;&#xB3C4; &#xC804;&#xC2DC; protease &#xBCC0;&#xC885;&#xC758; &#xACF5;&#xD559;.

La selectividad exquisita y actividad catal&iacute;tica de las enzimas han sido moldeados por los efectos de la presi&oacute;n de selecci&oacute;n positiva y negativa durante el curso de la evoluci&oacute;n. En cambio, variantes de la enzima dise&ntilde;adas mediante t&eacute;cnicas de proyecci&oacute;n in vitro para aceptar nuevos sustratos t&iacute;picamente muestran un mayor grado de promiscuidad catal&iacute;tica y menor volumen de negocios total en comparaci&oacute;n con sus contrapartes naturales. Utilizando la pantalla bacteriana y citometr&iacute;a de flujo multipar&aacute;metro, hemos desarrollado una nueva metodolog&iacute;a para emular positiva y negativa presi&oacute;n selectiva in vitro para el aislamiento de variantes de la enzima con reactividad sustratos novela deseada, mientras que simult&aacute;neamente excepto con reactividad hacia sustratos no deseados. Proyecci&oacute;n de una gran biblioteca de mutantes al azar de la endopeptidasa de coli Escherichia OmpT condujo al aislamiento de una variante del enzima, 1.3.19, que troceados un enlace pept&iacute;dico de Ala-Arg en vez de los bonos de Arg-Arg preferido por la enzima WT. 1.3.19 Variante exhibi&oacute; mayor que tres millones-veces de selectividad (- Ala - Arg-/ - Arg - Arg-) y una eficacia catal&iacute;tica para Ala-Arg escote que es igual a la mostrada por el padre para el sustrato preferido, Arg-Arg. Un solo amino&aacute;cido Ser223Arg sustituci&oacute;n se mostr&oacute; totalmente recapitular las &uacute;nicas propiedades catal&iacute;ticas de la 1.3.19 variante. Estos resultados pueden explicarse al proponer que esta mutaci&oacute;n act&uacute;a para &quot;cambiar&quot; de la cadena lateral de Arg p normalmente se encuentra en p&eacute;ptidos de sustrato WT con la cadena lateral de 223Arg en el subsitio de s de OmpT.

Ingenier&iacute;a de variantes de la proteasa que exhiben alta actividad catal&iacute;tica y selectividad de substrato exquisito.

The egress of HIV particles from virus-infected cells is accomplished by the recruitment of proteins that normally mediate host cell endocytic functions. This process requires interaction of the HIV Gag protein with the host protein TSG101 (tumor susceptibility gene 101). Here, we report the use of a bacterial reverse two-hybrid system to identify cyclic peptides that interfere with the Gag-TSG101 interaction and the finding that a five amino acid peptide discovered by this approach can disrupt the interaction and consequently inhibit HIV egress. The inhibiting molecule, which was selected from a cyclic peptide library containing approximately 3.2 x 10(6) members, differs in primary sequence from the interacting sites of either TSG101 or Gag. Addition of cyclic peptide tagged with an HIV Tat sequence, which previously has been shown to enhance protein translocation across plasma membranes, to cultured human cells inhibited the production of virus-like particles (VLPs) by these cells (IC(50) of 7 microM), and this inhibition occurred in the absence of adverse affects on normal endocytic functions mediated by TSG101. A mutant Gag protein not dependent on TSG101 for release was unaffected by the cyclic peptide. Our findings, which suggest that interference with the TSG101-Gag interaction by cyclic peptides may be of practical use in the treatment of HIV infections, identify a specific cyclic peptide that reduces VLP release by this mechanism; they also demonstrate that the efficiency of interference with protein-protein interactions by cyclic peptides can be enhanced by tagging the peptides with translocation-promoting sequences. Collectively our results support the notion that small molecule therapeutics that inhibit specific interactions between viral and host proteins may have general applicability in antiviral therapy.

Inhibition of HIV budding by a genetically selected cyclic peptide targeting the Gag-TSG101 interaction.

Drug resistance associated with dihydrofolate reductase (DHFR) has emerged as a critical issue in the treatment of bacterial infections. In our efforts to understand the mechanism of a drug-resistant dihydrofolate reductase (DHFR) from a pathogenic bacterial source, we report the first kinetic characterization of Streptococcus pneumoniae DHFR (spDHFR) along with its X-ray structure. This study revealed that the kinetic properties of spDHFR were significantly different from those of Escherichia coli DHFR. The product (tetrahydrofolate) dissociation step that is the rate-limiting step in E. coli DHFR is significantly accelerated in spDHFR so that hydride transfer or a preceding step is rate-limiting. Comparison of the binding parameters of this enzyme to those of a mutant spDHFR (Sp9) confirmed that the Leu100 residue in spDHFR is the critical element for the trimethoprim (TMP) resistance. Steady-state kinetics exhibited a pH dependence in k(cat), which prompted us to elucidate the role of the new catalytic residue (His33) in the active site of spDHFR. Structural data of the Sp9 mutant in complex with NADPH and methotrexate confirmed the participation of His33 in a hydrogen bonding network involving a water molecule, the hydroxyl group of Thr119, and the carboxylate ion of Glu30. Sequence analysis of the DHFR superfamily revealed that the His residue is the major amino acid component at this position and is found mostly in pathogenic bacterial DHFRs. A mutation of Val100 to Leu demonstrated a steric clash of the leucine side chain with the side chains of Ile8 and Phe34, rationalizing weaker binding of trimethoprim to Leu100 DHFR. Understanding the role of specific amino acids in the active site coupled with detailed structural analysis will inform us on how to better design inhibitors targeting drug-resistant pathogenic bacterial DHFRs.

Kinetic and structural characterization of dihydrofolate reductase from Streptococcus pneumoniae.

Conformational dynamics play a key role in enzyme catalysis. Although protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of Escherichia coli dihydrofolate reductase that abrogates millisecond-time-scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond time scale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for design of novel protein catalysts.

A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis.

Molecular biosensors were developed and applied to measure individual sugars in biological mixtures such as bacterial culture broths. As the sensing units, four sugar-binding proteins (SBPs for allose, arabinose, ribose, and glucose) were selected from the Escherichia coli genome and connected to a cyan fluorescent protein and yellow fluorescent protein via dipeptide linkers (CFP-L-SBP-YFP). The putative sensors were randomized in the linker region (L) and then investigated with regard to the intensity of fluorescence resonance energy transfer on the binding of the respective sugars. As a result, four representatives were selected from each library and examined for their specificity using 16 available sugars. The apparent dissociation constants of the allose, arabinose, ribose, and glucose sensors were estimated to be 0.35, 0.36, 0.17, and 0.18 μM. Finally, the sugar sensors were applied to monitor the consumption rate of individual sugars in an E. coli culture broth. The individual sugar profiles exhibited a good correlation with those obtained using an HPLC method, confirming that the biosensors offer a rapid and easy-to-use method for monitoring individual sugars in mixed compositions.

Quantitative analyses of individual sugars in mixture using FRET-based biosensors.

The enzymes in the human de novo purine synthesis pathway were found to form a cellular complex, the purinosome, upon culturing cells in purine-depleted medium (An, S., Kumar R., Sheets, E. D., and Benkovic, S. J. (2008) Science 320, 103-106). Purinosome formation and dissociation were found to be modulated by several factors, including the microtubule network and cell signaling involving protein phosphorylation. To determine whether the pathway enzymes are in physical contact, we probed for the protein-protein interactions (PPIs) within the purinosome with a novel application of the Tango PPI reporter system (Barnea, G., Strapps, W., Herrada, G., Berman, Y., Ong, J., Kloss, B., Axel, R., and Lee, K. J. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 64-69). We found PPIs among all six enzymes within the pathway and evidence for a core involving the first three enzymes. We also captured purinosomes under both purine-rich and purine-depleted conditions. The results provide additional insights into the transient nature and topography of the purinosome.

Mapping protein-protein proximity in the purinosome.

Elucidation of the signal-transmission pathways between distant sites within proteins is of great importance in medical and bioengineering sciences. The use of optical methods to redesign protein functions is emerging as a general approach for the control of biological systems with high spatiotemporal precision. Here we report the detailed thermodynamic and kinetic characterization of novel chimeric light-regulated Tet repressor (TetR) switches in which light modulates the TetR function. Light absorbed by flavin mononucleotide (FMN) generates a signal that is transmitted to As-LOV and YtvA-LOV fused TetR proteins (LOV=light-oxygen-voltage), in which it alters the binding to tetracycline, the TetR ligand. The engineering of light-sensing protein modules with TetR is a valuable tool that deepens our understanding of the mechanism of signal transmission within proteins. In addition, the light-regulated changes of drug binding that we describe here suggest that engineered light-sensitive proteins may be used for the development of novel therapeutic strategies.

Light-regulated tetracycline binding to the Tet repressor.

Fluorescence resonance energy transfer (FRET)-based sensors transduce ligand recognition into a change in the fluorophore spectrum, as ligand binding alters the distance between and orientation of two fluorescent proteins. Here, we report a dramatic increase in the signal intensity of FRET-based sugar sensors with bacterial periplasmic binding proteins (PBPs) in the binding moiety, by increasing the analysis temperature, usually higher than 50°C. The increased signal intensity results from a sudden decrease in background signal at critical temperatures, while recovering the maximum FRET ratios in the presence of ligands. When tested with a maltose sensor using a maltose-binding protein as the binding moiety, the FRET ratio at the critical temperature, 55°C, was 17-fold higher than at ambient temperatures. Similar effects were observed using analogous sensors for allose, arabinose, and glucose, providing highly dynamic and quantitative ratio changes at the critical temperatures. The proposed mechanism underlying the signal improvement is thermal relaxation of the binding proteins at the critical temperature; this hypothesis was supported by the results of intrinsic tryptophan fluorescence and circular dichroism experiments. In summary, this study shows that the conformational relaxation of proteins under specific conditions can be leveraged for highly sensitive and rapid measurements of ligands using FRET-based sensors.

Ratiometric analyses at critical temperatures can magnify the signal intensity of FRET-based sugar sensors with periplasmic binding proteins.

YtvA, a photosensory LOV (light-oxygen-voltage) protein from Bacillus subtilis, exists as a dimer that previously appeared to undergo surprisingly small structural changes after light illumination compared with other light-sensing proteins. However, we now report that light induces significant structural perturbations in a series of YtvA-LOV domain derivatives in which the Jα helix has been truncated or replaced. Results from native gel analysis showed significant mobility changes in these derivatives after light illumination; YtvA-LOV without the Jα helix dimerized in the dark state but existed as a monomer in the light state. The absence of the Jα helix also affected the dark regeneration kinetics and the stability of the flavin mononucleotide (FMN) binding to its binding site. Our results demonstrate an alternative way of photo-induced signal propagation that leads to a bigger functional response through dimer/monomer conversions of the YtvA-LOV than the local disruption of Jα helix in the As-LOV domain.

Article	Year
High-throughput FACS method for directed evolution of substrate specificity. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 12824593	2003
Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15867160	2005
Inhibition of HIV budding by a genetically selected cyclic peptide targeting the Gag-TSG101 interaction. ACS chemical biology\| PubMed ID: 19053244	2008
Kinetic and structural characterization of dihydrofolate reductase from Streptococcus pneumoniae. Biochemistry\| PubMed ID: 19950924	2010
A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis. Science (New York, N.Y.)\| PubMed ID: 21474759	2011
Quantitative analyses of individual sugars in mixture using FRET-based biosensors. Biotechnology progress\| PubMed ID: 22753346	2012
Mapping protein-protein proximity in the purinosome. The Journal of biological chemistry\| PubMed ID: 22955281	2012
Light-regulated tetracycline binding to the Tet repressor. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 24478132	2014
Ratiometric analyses at critical temperatures can magnify the signal intensity of FRET-based sugar sensors with periplasmic binding proteins. Biosensors & bioelectronics\| PubMed ID: 25957075	2015
A critical element of the light-induced quaternary structural changes in YtvA-LOV. Protein science : a publication of the Protein Society\| PubMed ID: 26402155	2015

Jongsik Gam

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