isolation-chemical-characterization-lipid-from-gram-negative

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

The University of Texas at Austin

A structured triblock protein was designed to explore the potential of engineered peptides to function as high-performance ink dispersants and binders. The protein consists of three functional elements, including a pigment binding domain, a hydrophilic linker, and a printing surface binding domain. To construct such a chimeric protein, a carbon black binding peptide, FHENWPS, and a cellulose binding peptide, THKTSTQRLLAA, were identified from phage display libraries through biopanning, based on their strong and specific binding affinities to carbon black and cellulose. They were used as carbon black and cellulose binding domains, respectively, in a recombinant triblock protein. A linker sequence, PTPTPTPTPTPTPTPTPTPTPTP, was adapted from endoglucanase A of the bacterium Cellulomonas fimi, as a small, rigid, and hydrophilic interdomain linker. When incorporated into the triblock structure between the carbon black and cellulose binding sequences, the linker sufficiently isolates these two elements and allows dual binding activity. The structured triblock protein was shown to disperse carbon black particles and attach it to paper surfaces. Thus, the utility of structured proteins having useful dispersant and binding properties for digital printing inks was demonstrated.

A recombinant triblock protein polymer with dispersant and binding properties for digital printing.

&#x771F;&#x6838;&#x4F38;&#x957F;&#x56E0;&#x5B50; 2 &#x6FC0;&#x9176; (eEF-2 K) &#x662F;&#x7531; Ca &lt;sup&gt;2 +&lt;/sup&gt; &#x548C;&#x9499;&#x8C03;&#x86CB;&#x767D; (CaM) &#x89C4;&#x5B9A;&#x975E;&#x5178;&#x578B;&#x7684;&#x86CB;&#x767D;&#x6FC0;&#x9176;&#x3002;&#x5176;&#x552F;&#x4E00;&#x5DF2;&#x77E5;&#x886C;&#x5E95;&#x662F;&#x771F;&#x6838;&#x4F38;&#x957F;&#x56E0;&#x7D20; 2 (eEF-2&#xFF09;&#xFF0C;&#x5176;&#x78F7;&#x9178;&#x5316; eEF-2 K &#x963B;&#x788D;&#x5168;&#x7403;&#x86CB;&#x767D;&#x8D28;&#x7684;&#x5408;&#x6210;&#x3002;&#x5230;&#x76EE;&#x524D;&#x4E3A;&#x6B62;&#xFF0C;eEF-2 K &#x81EA;&#x78F7;&#x9178;&#x5316;&#x7684;&#x673A;&#x5236;&#x5B8C;&#x5168;&#x9610;&#x660E;&#x3002;&#x8C03;&#x67E5;&#x81EA;&#x78F7;&#x9178;&#x5316;&#x7684;&#x673A;&#x5236;&#xFF0C;&#x4EBA;&#x7C7B; eEF-2 K &#x662F; co-expressed &#x4E0E; &lambda;-&#x78F7;&#x9178;&#x9176;&#xFF0C;&#x5E76;&#x4ECE;&#x7EC6;&#x83CC;&#x4E2D;&#x4E09;&#x6B65;&#x534F;&#x8BAE;&#x4F7F;&#x7528;&#x9499;&#x8C03;&#x7D20;-&#x4EB2;&#x548C;&#x67F1;&#x7EAF;&#x5316;&#x3002;&#x7EAF;&#x51C0; eEF-2 K &#x5230; autophosphorylate &#x8BF1;&#x5BFC;&#x4E0E; Ca &lt;sup&gt;2 +&lt;/sup&gt; &#x5B75;&#x5316; / CaM MgATP &#x9762;&#x524D;&#x3002;&#x8D28;&#x8C31;&#x6CD5;&#x5206;&#x6790;&#x80F0;&#x9176;&#x6216; chymotryptic &#x7684;&#x591A;&#x80BD;&#x76D1;&#x6D4B;&#x81EA;&#x78F7;&#x9178;&#x5316;&#x8D85;&#x8FC7; 0-180 &#x5206;&#x949F;&#x3002;&#x4EE5;&#x4E0B;&#x4E94;&#x4E2A;&#x4E3B;&#x8981;&#x81EA;&#x78F7;&#x9178;&#x5316;&#x5730;&#x70B9;&#x88AB;&#x786E;&#x5B9A;&#x3001; &#x82CF;&#x6C28;&#x9178;-348&#x3001; &#x82CF;&#x6C28;&#x9178; 353&#x3001; Ser 445&#x3001; Ser 474 &#x548C; Ser 500&#x3002;&#x5728;&#x573A;&#x7684;&#x60C5;&#x51B5;&#x4E0B; Ca &lt;sup&gt;2 +&lt;/sup&gt; / CaM&#xFF0C;&#x9C81;&#x68D2;&#x78F7;&#x9178;&#x5316;&#x82CF;&#x6C28;&#x9178; 348 &#x6DFB;&#x52A0; MgATP &#x79D2;&#x5185;&#x53D1;&#x751F;&#x3002;&#x8BF1;&#x53D8;&#x80B2;&#x79CD;&#x7684;&#x7814;&#x7A76;&#x8868;&#x660E;&#xFF0C;&#x78F7;&#x9178;&#x5316;&#x82CF;&#x6C28;&#x9178; 348 &#x662F;&#x6240;&#x5FC5;&#x9700;&#x7684;&#x57FA;&#x677F; &#xFF08;eEF 2 &#x6216;&#x80BD;&#x57FA;&#x8D28;&#xFF09; &#x78F7;&#x9178;&#x5316;&#xFF0C;&#x4F46;&#x4E0D;&#x662F; self-phosphorylation&#x3002;&#x78F7;&#x9178;&#x5316; Ser 500 &#x843D;&#x540E;&#x4E8E;&#x78F7;&#x9178;&#x5316;&#x82CF;&#x6C28;&#x9178;-348&#xFF0C;&#x5E76;&#x4E0E;&#x9499;&#x72EC;&#x7ACB;&#x6D3B;&#x52A8;&#x7684; 2 K eEF &#x76F8;&#x5173;&#x8054;&#x3002;&#x7A81;&#x53D8;&#x7684; Ser 500 &#x5230; Asp&#xFF0C;&#x4F46;&#x4E0D;&#x662F; Ala &#x5448;&#x73B0; eEF-2 K &#x9499;&#x65E0;&#x5173;&#x3002;&#x5947;&#x602A;&#x7684;&#x662F;&#xFF0C;&#x8FD9;&#x79CD;&#x9499;&#x72EC;&#x7ACB;&#x6D3B;&#x52A8;&#x9700;&#x8981;&#x9499;&#x8C03;&#x7D20;&#x7684;&#x5B58;&#x5728;&#x3002;

&#x9499;/&#x9499;&#x8C03;&#x7D20;&#x523A;&#x6FC0;&#x82CF;&#x6C28;&#x9178; 348 &#x548C; Ser 500 &#x6765;&#x89C4;&#x7BA1;&#x5176;&#x6D3B;&#x52A8;&#x53CA;&#x9499;&#x4F9D;&#x8D56; 2 &#x6FC0;&#x9176;&#x4F38;&#x957F;&#x56E0;&#x7D20;&#x81EA;&#x78F7;&#x9178;&#x5316;&#x3002;

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca2+ and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was co-expressed with Î»-phosphatase, and purified from bacteria in a three-step protocol using a calmodulin-affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca2+/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 minutes. The following five major autophosphorylation sites were identified, Thr-348, Thr-353, Ser-445, Ser-474 and Ser-500. In the presence of Ca2+/CaM, robust phosphorylation of Thr-348 occurs within seconds of adding MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348, and is associated with calcium-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K calcium-independent. Surprisingly, this calcium-independent activity requires the presence of calmodulin.

Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence.

Kinase de facteur 2 &eacute;longation eucaryote (2 K - Fee) est une kinase atypique r&eacute;gul&eacute;e de Ca &lt;sup&gt;2 +&lt;/sup&gt; et la calmoduline (CaM). Son seul substrat connu est facteur d&#39;&eacute;longation eucaryote 2 (eEF-2), dont la phosphorylation par eEF - 2 K emp&ecirc;che la synth&egrave;se prot&eacute;ique globale. A ce jour, le m&eacute;canisme de l&#39;eEF - 2 K autophosphorylation n&#39;a pas &eacute;t&eacute; compl&egrave;tement &eacute;lucid&eacute;. Pour &eacute;tudier le m&eacute;canisme d&#39;autophosphorylation, humain eEF - 2 K est co-exprim&eacute;e avec &lambda;-phosphatase et purifi&eacute;e &agrave; partir de bact&eacute;ries dans un protocole de trois &eacute;tapes &agrave; l&#39;aide d&#39;une colonne de la calmoduline-affinit&eacute;. Purifi&eacute;e eEF - 2 K a &eacute;t&eacute; amen&eacute;e &agrave; autophosphorylate par incubation avec Ca &lt;sup&gt;2 +&lt;/sup&gt; / CaM en pr&eacute;sence de MgATP. Analyse des peptides trypsiques ou chymotrypsiques par spectrom&eacute;trie de masse surveill&eacute; l&#39;autophosphorylation de plus de 0 &agrave; 180 minutes. Les cinq sites d&#39;autophosphorylation majeurs suivants ont &eacute;t&eacute; identifi&eacute;s, Thr-348, Thr-353, Ser-445, Ser-474 et Ser-500. En pr&eacute;sence de Ca &lt;sup&gt;2 +&lt;/sup&gt; / CaM, la phosphorylation de Thr-348 robuste se produit quelques secondes de l&#39;ajout de MgATP. Les &eacute;tudes de mutagen&egrave;se sugg&egrave;rent que la phosphorylation de Thr-348 est n&eacute;cessaire pour la phosphorylation du substrat (eEF-2 ou un substrat peptidique), mais pas self-phosphorylation. La phosphorylation de Ser-500 est &agrave; la tra&icirc;ne de la phosphorylation de Thr-348 et est associ&eacute;e &agrave; l&#39;activit&eacute; ind&eacute;pendante du calcium d&#39;eEF - 2 K. Mutation de Ser-500 &agrave; Asp, mais pas Ala, rend ind&eacute;pendant du calcium eEF - 2 K. &Eacute;tonnamment, cette activit&eacute; ind&eacute;pendante du calcium n&eacute;cessite la pr&eacute;sence de la calmoduline.

Calcium/calmoduline stimule l&#39;Autophosphorylation d&#39;&eacute;longation facteur 2 Kinase sur Thr-348 et Ser-500 de r&eacute;guler son activit&eacute; et la d&eacute;pendance calcique.

Eukaryotische Dehnung Faktor 2 Kinase (eEF - 2 K) ist eine atypische Proteinkinase von Ca &lt;sup&gt;2 +&lt;/sup&gt; und Calmodulin (CaM) geregelt. Die einzige bekannte Substrat ist eukaryotischen Dehnung Faktor 2 (eEF-2), dessen Phosphorylierung von eEF - 2 K global Proteinsynthese hemmt. Bis heute ist der Mechanismus der eEF - 2K Autophosphorylierung nicht vollst&auml;ndig gekl&auml;rt. Um den Mechanismus der Autophosphorylierung zu untersuchen, wurde menschliche eEF - 2K co-expressed mit &lambda;-Phosphatase und gereinigt von Bakterien in einem drei-Stufen-Protokoll mit einem Calmodulin-Affinit&auml;tss&auml;ule. Gereinigte eEF - 2K wurde zu Autophosphorylate induziert, durch Inkubation mit Ca &lt;sup&gt;2 +&lt;/sup&gt; / CaM in Anwesenheit von MgATP. Analyse von tryptic oder chymotryptic Peptide durch Massenspektrometrie &uuml;berwacht die Autophosphorylierung &uuml;ber 0-180 Minuten. Die folgenden f&uuml;nf gro&szlig;en Autophosphorylierung Websites wurden festgestellt, Thr-348, Thr-353, Ser-445, Ser-474 und Ser-500. In Anwesenheit von Ca &lt;sup&gt;2 +&lt;/sup&gt; / CaM, robuste Phosphorylierung von Thr-348 tritt innerhalb von Sekunden MgATP hinzuf&uuml;gen. Mutagenese Studien deuten darauf hin, dass die Phosphorylierung von Thr-348 ist erforderlich f&uuml;r die Phosphorylierung von Substrat (eEF-2 oder einem Peptid-Substrat), aber nicht Self-phosphorylation. Phosphorylierung von Ser-500 hinkt die Phosphorylierung von Thr-348 und Kalzium-unabh&auml;ngige Aktivit&auml;t von eEF - 2 K zugeordnet ist. Mutation des Ser-500 zu Asp, aber nicht Ala, rendert eEF - 2K Kalzium-unabh&auml;ngig. &Uuml;berraschenderweise erfordert diese Kalzium-unabh&auml;ngige T&auml;tigkeit die Anwesenheit von Calmodulin.

Calcium/Calmodulin stimuliert die Autophosphorylierung der Dehnung Faktor 2 Kinase Thr-348 und Ser-500 auf, ihre Aktivit&auml;ten und Calcium-Abh&auml;ngigkeit zu regulieren.

Chinasi fattore 2 di allungamento eucariotico (eEF-2K) &egrave; una chinasi di proteina atipico regolata da Ca &lt;sup&gt;2++&lt;/sup&gt; e calmodulina (CaM). Suo solo substrato conosciuta &egrave; fattore di allungamento eucariotico 2 (eEF-2), cui la fosforilazione di eEF-2 K impedisce la sintesi proteica globale. Ad oggi, il meccanismo di autophosphorylation K eEF-2 non &egrave; stata completamente chiarito. Per studiare il meccanismo di autophosphorylation, umano eEF-2 K era co-expressed con &lambda;-fosfatasi e purificata da batteri in un three-step protocollo utilizzando una colonna di affinit&agrave; calmodulina. Purificato eEF-2 K fu indotto a autophosphorylate di incubazione con Ca &lt;sup&gt;2++&lt;/sup&gt; / CaM in presenza di MgATP. Analisi di peptidi triptico o chymotryptic di spettrometria di massa monitorato il autophosphorylation oltre 0-180 minuti. I seguenti cinque principali autophosphorylation siti sono stati identificati, Thr-348, Thr-353, Ser-445, Ser-474 e Ser-500. In presenza di Ca &lt;sup&gt;2++&lt;/sup&gt; / CaM, robusta fosforilazione della Thr-348 si verifica in pochi secondi di aggiunta MgATP. Mutagenesi gli studi suggeriscono che la fosforilazione della Thr-348 &egrave; richiesto per la fosforilazione del substrato (eEF-2 o un substrato peptide), ma non self-phosphorylation. Fosforilazione della Ser-500 in ritardo rispetto la fosforilazione della Thr-348 ed &egrave; associata con attivit&agrave; di calcio-indipendenti di eEF-2 K. Mutazione di Ser-500 per Asp, ma non Ala, esegue il rendering di eEF-2 K calcio-indipendenti. Sorprendentemente, questa attivit&agrave; calcio-indipendente richiede la presenza della calmodulina.

Calcio/calmodulina stimola il Autophosphorylation di allungamento Factor 2 chinasi Thr-348 e Ser-500 a regolare la sua attivit&agrave; e la dipendenza dal calcio.

&#x771F;&#x6838;&#x4F38;&#x9577;&#x56E0;&#x5B50; 2 &#x30AD;&#x30CA;&#x30FC;&#x30BC; (eEF 2 K) Ca &lt;sup&gt;2++&lt;/sup&gt; &#x304A;&#x3088;&#x3073;&#x30AB;&#x30EB;&#x30E2;&#x30C7;&#x30E5;&#x30EA;&#x30F3; &#xFF08;CaM&#xFF09; &#x306B;&#x3088;&#x3063;&#x3066;&#x898F;&#x5236;&#x3055;&#x308C;&#x3066;&#x3001;&#x975E;&#x5B9A;&#x578B;&#x306E;&#x86CB;&#x767D;&#x8CEA;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x552F;&#x4E00;&#x306E;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x305F;&#x57FA;&#x677F;&#x771F;&#x6838;&#x4F38;&#x9577;&#x56E0;&#x5B50; 2 (eEF-2&#xFF09;&#x3001;&#x305D;&#x306E; eEF 2 K &#x306B;&#x3088;&#x308B;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30B0;&#x30ED;&#x30FC;&#x30D0;&#x30EB; &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5408;&#x6210;&#x963B;&#x5BB3;&#x3067;&#x3059;&#x3002;&#x65E5;&#x4ED8;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;eEF 2 K &#x81EA;&#x5DF1;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x5B8C;&#x5168;&#x306B;&#x89E3;&#x660E;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;&#x81EA;&#x5DF1;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x6A5F;&#x69CB;&#x3092;&#x89E3;&#x660E;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x4EBA;&#x9593; eEF 2 K &lambda; &#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30BF;&#x30FC;&#x30BC;&#x3068;&#x5171;&#x540C;&#x3001;&#x30AB;&#x30EB;&#x30E2;&#x30B8;&#x30E5;&#x30EA;&#x30F3;&#x89AA;&#x548C;&#x6027;&#x30AB;&#x30E9;&#x30E0;&#x3092;&#x7528;&#x3044;&#x305F; 3 &#x6BB5;&#x968E;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30FC;&#x30EB;&#x306E;&#x7D30;&#x83CC;&#x304B;&#x3089;&#x7CBE;&#x88FD;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x7CBE;&#x88FD; eEF 2 K autophosphorylate &#x306B;&#x30AB;&#x30EA;&#x30D5;&#x30A9;&#x30EB;&#x30CB;&#x30A2;&#x5DDE; &lt;sup&gt;2++&lt;/sup&gt; &#x3068;&#x5B75;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066;&#x8A98;&#x8D77;&#x3055;&#x308C;&#x305F;/&#x30AB;&#x30E0; MgATP &#x5B58;&#x5728;&#x4E0B;&#x3067;&#x3002;&#x30C8;&#x30EA;&#x30D7;&#x30B7;&#x30F3;&#x307E;&#x305F;&#x306F; chymotryptic &#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x3092;&#x8CEA;&#x91CF;&#x5206;&#x6790;&#x6CD5;&#x306B;&#x3088;&#x308B;&#x5206;&#x6790;&#x76E3;&#x8996;&#x81EA;&#x5DF1;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x4EE5;&#x4E0A; 0-180 &#x5206;&#x3002;&#x6B21;&#x306E; 5 &#x3064;&#x306E;&#x4E3B;&#x8981;&#x306A;&#x81EA;&#x5DF1;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30B5;&#x30A4;&#x30C8;&#x8B58;&#x5225;&#x3055;&#x308C;&#x305F;&#x3001;Thr 348 Thr 353, Ser 445, Ser 474 Ser 500.Ca &lt;sup&gt;2++&lt;/sup&gt; &#x306E;&#x5B58;&#x5728;&#x4E0B;&#x3067;/&#x30AB;&#x30E0;&#x3001;&#x5805;&#x7262;&#x306A;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30B9;&#x30EC;&#x30AA;&#x30CB;&#x30F3; 348 &#x306E; MgATP &#x8FFD;&#x52A0;&#x306E;&#x79D2;&#x4EE5;&#x5185;&#x306B;&#x767A;&#x751F;&#x3057;&#x307E;&#x3059;&#x3002;&#x7A81;&#x7136;&#x5909;&#x7570;&#x8A98;&#x767A;&#x306E;&#x7814;&#x7A76;&#x793A;&#x5506;&#x305D;&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30B9;&#x30EC;&#x30AA;&#x30CB;&#x30F3; 348 &#x57FA;&#x677F; &#xFF08;eEF 2 &#x307E;&#x305F;&#x306F;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x57FA;&#x8CEA;&#xFF09; &#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x304C;&#x306A;&#x3044; self-phosphorylation &#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;Ser 500 &#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30B9;&#x30EC;&#x30AA;&#x30CB;&#x30F3; 348 &#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306E;&#x5F8C;&#x308D;&#x306B;&#x9045;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;eEF 2 K &#x306E;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x975E;&#x4F9D;&#x5B58;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;Ser-500 &#x306B; Asp&#x3001;&#x3057;&#x304B;&#x3057;&#x306A;&#x3044; Ala &#x306E;&#x5909;&#x7570; eEF 2 K &#x306F;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x975E;&#x4F9D;&#x5B58;&#x3092;&#x30EC;&#x30F3;&#x30C0;&#x30EA;&#x30F3;&#x30B0;&#x3057;&#x307E;&#x3059;&#x3002;&#x9A5A;&#x3044;&#x305F;&#x3053;&#x3068;&#x306B;&#x3001;&#x3053;&#x306E;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x72EC;&#x7ACB;&#x6D3B;&#x52D5;&#x30AB;&#x30EB;&#x30E2;&#x30C7;&#x30E5;&#x30EA;&#x30F3;&#x306E;&#x5B58;&#x5728;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;

&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;/&#x30AB;&#x30EB;&#x30E2;&#x30B8;&#x30E5;&#x30EA;&#x30F3;&#x3092;&#x523A;&#x6FC0;&#x3059;&#x308B; Thr 348 &#x3068; Ser 500 &#x305D;&#x306E;&#x6D3B;&#x52D5;&#x3068;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x4F9D;&#x5B58;&#x6027;&#x3092;&#x898F;&#x5236;&#x3059;&#x308B;&#x4F38;&#x9577;&#x56E0;&#x5B50; 2 &#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306E;&#x81EA;&#x5DF1;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3002;

&#xC9C4; &#xD575; &#xC2E0;&#xC7A5; &#xC778;&#xC790; 2 &#xD0A4; (eEF-2 K)&#xB294; &#xBE44;&#xC815;&#xD615; &#xB2E8;&#xBC31;&#xC9C8; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; Ca &lt;sup&gt;2 +&lt;/sup&gt;&#xC640; calmodulin (CaM)&#xC5D0; &#xC758;&#xD574; &#xADDC;&#xC81C; &#xC774;&#xB2E4;. &#xADF8;&#xAC83;&#xC758; &#xC720;&#xC77C;&#xD55C; &#xC54C;&#xB824;&#xC9C4;&#xB41C; &#xAE30;&#xD310;&#xC740; &#xC9C4; &#xD575; &#xC2E0;&#xC7A5; &#xC694;&#xC778; 2 (eEF-2), &#xB204;&#xAD6C;&#xC758; &#xC778; &#xC0B0;&#xD654; eEF-2 &#xCF00;&#xC774; &#xAE00;&#xB85C;&#xBC8C; &#xB2E8;&#xBC31;&#xC9C8; &#xD569;&#xC131;&#xC744; &#xBC29;&#xD574; &#xD55C;&#xB2E4;. &#xC9C0;&#xAE08;&#xAE4C;&#xC9C0; eEF K autophosphorylation&#xC758; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC740; &#xD558;&#xC9C0; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4; &#xC644;&#xC804;&#xD788; &#xD574;&#xBA85;. Autophosphorylation &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC870;&#xC0AC; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC778;&#xAC04;&#xC758; eEF-2 K &lambda;-&#xC778;&#xC0B0; &#xAC00;&#xC218;&#xBD84;&#xD574; &#xD6A8;&#xC18C;&#xC640; co-expressed &#xB418;&#xC5C8;&#xACE0; calmodulin &#xC120;&#xD638;&#xB3C4; &#xC5F4;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; 3 &#xB2E8;&#xACC4; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;&#xC5D0; &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xC5D0;&#xC11C; &#xC815;&#xD654;. &#xC815;&#xD654; eEF-2k autophosphorylate&#xC744; Ca &lt;sup&gt;2 +&lt;/sup&gt; &#xBD80; &#xD654;&#xC5D0; &#xC758;&#xD574; &#xC720;&#xB3C4; / &#xCEA0; MgATP &#xC874;&#xC7AC;. &#xC9C8;&#xB7C9; &#xBD84;&#xC11D;, tryptic &#xB610;&#xB294; chymotryptic &#xD3A9; &#xD2F0; &#xB4DC; &#xBD84;&#xC11D; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; autophosphorylation &#xC774;&#xC0C1; 0-180 &#xBD84;. &#xB2E4;&#xC74C;&#xACFC; &#xAC19;&#xC740; 5 &#xAC1C;&#xC758; &#xC8FC;&#xC694; autophosphorylation &#xC0AC;&#xC774;&#xD2B8; &#xD655;&#xC778;, Thr-348, Thr-353, Ser-445, Ser-474&#xC640; Ser-500 &#xC774;&#xC5C8;&#xB2E4;. Ca &lt;sup&gt;2 +&lt;/sup&gt;&#xC758; &#xC874;&#xC7AC; / &#xCEA0;, Thr-348&#xC758; &#xAC15;&#xB825;&#xD55C; &#xC778; &#xC0B0;&#xD654; MgATP &#xCD94;&#xAC00;&#xC758; &#xCD08; &#xC774;&#xB0B4;&#xC5D0; &#xBC1C;&#xC0DD; &#xD569;&#xB2C8;&#xB2E4;. Mutagenesis &#xC5F0;&#xAD6C;&#xC758; &#xC778; &#xC0B0;&#xD654; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4; Thr-348&#xB294; &#xAE30;&#xD310; (eEF 2 &#xB610;&#xB294; &#xD3A9; &#xD2F0; &#xB4DC; &#xAE30;&#xC9C8;) &#xC778; &#xC0B0;&#xD654;, &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; self-phosphorylation&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. Ser-500&#xC758; &#xC778; &#xC0B0;&#xD654; &#xB4A4;&#xC9C4; &#xAC83;&#xC73C;&#xB85C; Thr-348&#xC758; &#xC778; &#xC0B0;&#xD654;&#xC640; &#xCE7C;&#xC298; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xD65C;&#xB3D9; eEF-2 K &#xAD00;&#xB828;. Asp, &#xD558;&#xC9C0;&#xB9CC; Ala &#xB80C;&#xB354;&#xB9C1; eEF K &#xCE7C;&#xC298; &#xB3C5;&#xB9BD;&#xC801; Ser-500&#xC758; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;. &#xB180;&#xB78D;&#xAC8C;&#xB3C4;,&#xC774; &#xCE7C;&#xC298; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xD65C;&#xB3D9; calmodulin&#xC758; &#xC874;&#xC7AC;&#xB97C; &#xD544;&#xC694;&#xD569;&#xB2C8;&#xB2E4;.

&#xC790;&#xADF9; &#xD558;&#xB294; &#xCE7C;&#xC298;/Calmodulin&#xB294; Autophosphorylation&#xC758; &#xC5F0;&#xC2E0; &#xC728; &#xC694;&#xC18C; 2 &#xD0A4;&#xC758; &#xD65C;&#xB3D9; &#xBC0F; &#xCE7C;&#xC298; &#xC758;&#xC874;&#xC131; Thr-348 Ser-500&#xC5D0;.

Quinasa de factor 2 de elongaci&oacute;n eucariota (eEF - 2 K) es un anormal de la prote&iacute;na quinasa regulada por Ca &lt;sup&gt;2 +&lt;/sup&gt; y calmodulina (CaM). Su &uacute;nico sustrato conocido es factor 2 (eEF-2), cuya fosforilaci&oacute;n por eEF - 2 K impide la s&iacute;ntesis de prote&iacute;nas global de elongaci&oacute;n eucariota. Hasta la fecha, el mecanismo de eEF - 2 K autophosphorylation no se ha aclarado completamente. Para investigar el mecanismo de autophosphorylation, humano eEF - 2 K fue co-expressed con &lambda;-fosfatasa y purificado de la bacteria en un protocolo de tres pasos usando una columna de afinidad calmodulina. Purificada eEF - 2 K fue inducida a autophosphorylate por la incubaci&oacute;n con Ca &lt;sup&gt;2 +&lt;/sup&gt; / CaM en presencia de MgATP. Analizando los p&eacute;ptidos tr&iacute;pticos o chymotryptic por espectrometr&iacute;a de masa monitoreado el autophosphorylation m&aacute;s de 0-180 minutos. Los siguientes cinco autophosphorylation principales sitios fueron identificados, Thr-348, Thr-353, Ser-445, 474-Ser y Ser-500. En presencia de Ca &lt;sup&gt;2 +&lt;/sup&gt; / CaM, fosforilaci&oacute;n robusta de Thr-348 ocurre dentro de segundos de a&ntilde;adir MgATP. Mutag&eacute;nesis estudios sugieren que la fosforilaci&oacute;n de Thr-348 es necesaria para la fosforilaci&oacute;n de sustrato (eEF-2 o un sustrato de p&eacute;ptido), pero no self-phosphorylation. Fosforilaci&oacute;n de Ser-500 va a la zaga de la fosforilaci&oacute;n de Thr-348 y se asocia a la actividad independiente de calcio de eEF - 2 K. Mutaci&oacute;n de Ser-500 a Asp, pero no Ala, rinde eEF - 2 K calcio-independiente. Sorprendentemente, esta actividad independiente de calcio requiere la presencia de calmodulina.

Calcio/calmodulina estimula el Autophosphorylation de alargamiento Factor 2 cinasa en Thr-348 y Ser-500 para regular su actividad y la dependencia de calcio.

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C.

Mapping protein surface accessibility via an electron transfer dissociation selectively cleavable hydrazone probe.

Campylobacter jejuni is a natural commensal of the avian intestinal tract. However, the bacterium is also the leading cause of acute bacterial diarrhea worldwide and is implicated in development of Guillain-BarrÃ© syndrome. Like many bacterial pathogens, C. jejuni assembles complex surface structures that interface with the surrounding environment and are involved in pathogenesis. Recent work in C. jejuni identified a gene encoding a novel phosphoethanolamine (pEtN) transferase, EptC (Cj0256), that plays a promiscuous role in modifying the flagellar rod protein, FlgG; the lipid A domain of lipooligosaccharide (LOS); and several N-linked glycans. In this work, we report that EptC catalyzes the addition of pEtN to the first heptose sugar of the inner core oligosaccharide of LOS, a fourth enzymatic target. We also examine the role pEtN modification plays in circumventing detection and/or killing by host defenses. Specifically, we show that modification of C. jejuni lipid A with pEtN results in increased recognition by the human Toll-like receptor 4-myeloid differentiation factor 2 (hTLR4-MD2) complex, along with providing resistance to relevant mammalian and avian antimicrobial peptides (i.e., defensins). We also confirm the inability of aberrant forms of LOS to activate Toll-like receptor 2 (TLR2). Most exciting, we demonstrate that strains lacking eptC show decreased commensal colonization of chick ceca and reduced colonization of BALB/cByJ mice compared to wild-type strains. Our results indicate that modification of surface structures with pEtN by EptC is key to its ability to promote commensalism in an avian host and to survive in the mammalian gastrointestinal environment.

EptC of Campylobacter jejuni mediates phenotypes involved in host interactions and virulence.

Since 2008 Massachusetts has had universal health insurance with an individual mandate. As a result, only about 3% of the population is uninsured. However, patients who use behavioral health services are uninsured at much higher rates. This 2011 study sought to understand reasons for the discrepancy and identify approaches to reduce disenrollment and sustain coverage.

Sustaining enrollment in health insurance for vulnerable populations: lessons from Massachusetts.

A chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating structures of proteins and protein complexes is reported, as demonstrated for lysozyme and beta-lactoglobulin with and without bound ligands. The chemical probe, NN, incorporates a UV chromophore that endows peptides with high cross sections at 351 nm, a wavelength not absorbed by unmodified peptides. Thus, NN-modified peptides can readily be differentiated from nonmodified peptides in complex tryptic digests created upon proteolysis of proteins after their exposure to the NN chemical probe. The NN chemical probe also affords two diagnostic reporter ions detected upon UVPD of the NN-modified peptide that provides a facile method for the identification of NN peptides within complex mixtures. Quantitation of the modified and unmodified peptides allows estimation of the surface accessibilities of lysine residues based on their relative reactivities with the NN chemical probe.

Article	Year
A recombinant triblock protein polymer with dispersant and binding properties for digital printing. Biopolymers\| PubMed ID: 17972282	2008
Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence. Biochemistry\| PubMed ID: 22329831	2012
Mapping protein surface accessibility via an electron transfer dissociation selectively cleavable hydrazone probe. Molecular & cellular proteomics : MCP\| PubMed ID: 22393264	2012
EptC of Campylobacter jejuni mediates phenotypes involved in host interactions and virulence. Infection and immunity\| PubMed ID: 23184526	2013
Sustaining enrollment in health insurance for vulnerable populations: lessons from Massachusetts. Psychiatric services (Washington, D.C.)\| PubMed ID: 23319011	2013
Chromogenic Chemical Probe for Protein Structural Characterization via Ultraviolet Photodissociation Mass Spectrometry. Analytical chemistry\| PubMed ID: 23855605	2013

John P. O'Brien

Biography

JoVE Journal Publications

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