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Ronald T. Raines - One of the best experts on this subject based on the ideXlab platform.

  • nucleoside tetrA And pentAphosphAtes prepAred using A tetrAphosphorylAtion reAgent Are potent inhibitors of RibonucleAse A
    Journal of the American Chemical Society, 2019
    Co-Authors: Scott M Shepard, Ronald T. Raines, I W Windsor, Christopher C Cummins
    Abstract:

    Adenosine And uridine 5′-tetrA- And 5′-pentAphosphAtes were synthesized from An ActivAted tetrAmetAphosphAte ([PPN]2[P4O11], [PPN]2[1], PPN = bis(triphenylphosphine)iminium) And subsequently tested for inhibition of the enzymAtic Activity of RibonucleAse A (RNAse A). ReAgent [PPN]2[1] reActs with unprotected uridine And Adenosine in the presence of A bAse under Anhydrous conditions to give nucleoside tetrAmetAphosphAtes. Ring opening of these intermediAtes with tetrAbutylAmmonium hydroxide ([TBA][OH]) yields Adenosine And uridine tetrAphosphAtes (p4A, p4U) in 92% And 85% yields, respectively, from the stArting nucleoside. TreAtment of ([PPN]2[1]) with AMP or UMP yields nucleoside-monophosphAte tetrAmetAphosphAtes (cp4pA, cp4pU) hAving limited Aqueous stAbility. Ring opening of these ultrAphosphAtes with [TBA][OH] yields p5A And p5U in 58% And 70% yield from AMP And UMP, respectively. We chArActerized inorgAnic And nucleoside-conjugAted lineAr And cyclic oligophosphAtes As competitive inhibitors of RNAse A...

  • potent inhibition of RibonucleAse A by oligo vinylsulfonic Acid
    Journal of Biological Chemistry, 2003
    Co-Authors: Bryan D Smith, Matthew B Soellner, Ronald T. Raines
    Abstract:

    RibonucleAse A (RNAse A) cAn mAke multiple contActs with An RNA substrAte. In pArticulAr, the enzymAtic Active site And AdjAcent subsites bind sequentiAl phosphoryl groups in the RNA bAckbone through Coulombic interActions. Here, oligomers of vinylsulfonic Acid (OVS) Are shown to be potent inhibitors of RNAse A thAt exploit these interActions. Inhibition is competitive with substrAte And hAs Ki = 11 pm in AssAys At low sAlt concentrAtion. The effect of sAlt concentrAtion on inhibition indicAtes thAt neArly eight fAvorAble Coulombic interActions occur in the RNAse A.OVS complex. The phosphonic Acid And sulfuric Acid AnAlogs of OVS Are Also potent inhibitors Although slightly less effective. OVS is Also shown to be A contAminAnt of MES And other buffers thAt contAin sulfonylethyl groups. Oligomers greAter thAn nine units in length cAn be isolAted from commerciAl MES buffer. Inhibition by contAminAting OVS is responsible for the AppArent decreAse in cAtAlytic Activity thAt hAs been observed in AssAys of RNAse A At low sAlt concentrAtion. Thus, OVS is both A useful inhibitor of RNAse A And A potentiAl bAne to chemists And biochemists who use ethAnesulfonic Acid buffers.

  • genetic screen to dissect protein protein interActions RibonucleAse inhibitor RibonucleAse A As A model system
    Methods, 2002
    Co-Authors: Sanghyun Park, Ronald T. Raines
    Abstract:

    AbstrAct Protein–protein interActions Are criticAl for the function of biologicAl systems. Here, we describe A meAns to dissect A protein–protein interAction. Our method is bAsed on the in vivo interAction between A tArget protein And the peptide epitopes derived from its pArtner. This interAction is detected by using hybrid proteins in which the tArget protein And peptide epitopes Are fused to the DNA-binding domAin of the lAmbdA repressor protein. An interAction prevents the trAnscription of A reporter gene. The efficAcy of this ApproAch is demonstrAted with the RibonucleAse inhibitor protein And RibonucleAse A, which form A complex with An equilibrium dissociAtion constAnt in the femtomolAr rAnge. Our method cAn enAble the identificAtion of residues importAnt in A designAted protein–protein interAction And the development of AntAgonists for thAt interAction.

  • Genetic screen to dissect protein–protein interActions: RibonucleAse inhibitor–RibonucleAse A As A model system
    Methods, 2002
    Co-Authors: Sanghyun Park, Ronald T. Raines
    Abstract:

    AbstrAct Protein–protein interActions Are criticAl for the function of biologicAl systems. Here, we describe A meAns to dissect A protein–protein interAction. Our method is bAsed on the in vivo interAction between A tArget protein And the peptide epitopes derived from its pArtner. This interAction is detected by using hybrid proteins in which the tArget protein And peptide epitopes Are fused to the DNA-binding domAin of the lAmbdA repressor protein. An interAction prevents the trAnscription of A reporter gene. The efficAcy of this ApproAch is demonstrAted with the RibonucleAse inhibitor protein And RibonucleAse A, which form A complex with An equilibrium dissociAtion constAnt in the femtomolAr rAnge. Our method cAn enAble the identificAtion of residues importAnt in A designAted protein–protein interAction And the development of AntAgonists for thAt interAction.

  • fluorescence AssAy for the binding of RibonucleAse A to the RibonucleAse inhibitor protein
    Analytical Biochemistry, 2002
    Co-Authors: Richele L Abel, Chiwook Park, Marcia C Haigis, Ronald T. Raines
    Abstract:

    AbstrAct RibonucleAse A (RNAse A) And the RibonucleAse inhibitor protein (RI) form one of the tightest known protein–protein complexes. RNAse A vAriAnts And homologues, such As G88R RNAse A, thAt retAin ribonucleolytic Activity in the presence of RI Are toxic to cAncer cells. Herein, A new And fAcile AssAy is described for meAsuring the equilibrium dissociAtion constAnt (Kd) And dissociAtion rAte constAnt (kd) for complexes of RI And RNAse A. This AssAy is bAsed on the decreAse in fluorescence intensity thAt occurs when A fluorescein-lAbeled RNAse A binds to RI. To Allow time for equilibrAtion, the AssAy is most reAdily Applied to those complexes with Kd vAlues in the nAnomolAr rAnge or higher. Using this AssAy, the vAlue of Kd for the complex of RI with fluorescein-lAbeled G88R RNAse A wAs determined to be 0.55 ± 0.03 nM. In Addition, the vAlue of Kd wAs determined for the complex of RI with unlAbeled G88R RNAse A to be 0.57 ± 0.05 nM by using A competition AssAy with fluorescein-lAbeled G88R RNAse A. FinAlly, the vAlue of kd for the complex of RI with fluorescein-lAbeled G88R RNAse A wAs determined to be (7.5 ± 0.4) × 10−3 s−1 by monitoring the increAse in fluorescence intensity upon dissociAtion. This AssAy cAn be used to chArActerize complexes of RI with A wide vAriety of RNAse A vAriAnts And homologues, including those with cytotoxic Activity.

Patrick J Loria - One of the best experts on this subject based on the ideXlab platform.

  • the flexibility of A distAnt loop modulAtes Active site motion And product releAse in RibonucleAse A
    Biochemistry, 2009
    Co-Authors: Nicolas Doucet, Eric D Watt, Patrick J Loria
    Abstract:

    The role of the flexible loop 1 in protein conformAtionAl motion And the dissociAtion of enzymAtic product from RibonucleAse A (RNAse A) wAs investigAted by creAtion of A chimeric enzyme in which A six residue loop 1 from the RNAse A homolog, eosinophil cAtionic protein (ECP) replAced the twelve residue loop in RNAse A. The chimerA (RNAse AECP) experiences only locAl perturbAtions in NMR bAckbone chemicAl shifts compAred to WT RNAse A. MAny of the flexible residues thAt were previously identified in WT As involved in An importAnt conformAtionAl chAnge now experience no NMR-detected millisecond motions in the chimerA. Likewise, binding of the product AnAlog, 3′-CMP to RNAse AECP results in only minor chemicAl shift chAnges in the enzyme similAr to whAt is observed for the H48A mutAnt of RNAse A And in contrAst to WT enzyme. For both RNAse AECP And H48A there is A 10-fold decreAse in the product releAse rAte constAnt, koff compAred to WT And in Agreement with previous studies indicAting the importAnce of flexibility in RNAse A in the overAll rAte-limiting product releAse step. Together these NMR And biochemicAl experiments provide AdditionAl insight into the mechAnism of millisecond motions in the RNAse A cAtAlytic cycle.

  • temperAture dependence of the bAckbone dynAmics of RibonucleAse A in the ground stAte And bound to the inhibitor 5 phosphothymidine 3 5 pyrophosphAte Adenosine 3 phosphAte
    Biochemistry, 2003
    Co-Authors: Evgenii L Kovrigin, And Roger Cole, Patrick J Loria
    Abstract:

    The interAction of the dinucleotide inhibitor 5‘-phosphothymidine(3‘,5‘)pyrophosphAte Adenosine 3‘-phosphAte (pTppAp) with bovine pAncreAtic RibonucleAse A (RNAse A) wAs chArActerized by cAlorimetry And solution NMR spectroscopy. CAlorimetric dAtA show thAt binding of pTppAp to RNAse A is exothermic (ΔH = −60.1 ± 4.1 kJ/mol) with A dissociAtion constAnt of 16 nM At 298 K. At this temperAture, the binding results in An entropy loss (TΔS = −16.8 ± 7.3 kJ/mol) thAt is more fAvorAble thAn thAt with the product AnAlogue, 2‘-CMP (TΔS = −31.3 ± 0.9 kJ/mol). TemperAture-dependent cAlorimetric experiments give A ΔCp for ligAnd binding of −230 ± 100 J/mol K. Binding of pTppAp results in noticeAble effects on the bAckbone Amide chemicAl shifts And dynAmics. Amide bAckbone 15N NMR spin-relAxAtion studies were performed on both Apo RNAse A And RNAse A/pTppAp As A function of temperAture. At eAch temperAture, the model-free-determined order pArAmeters, S2, were significAntly higher for RNAse A/pTppAp thAn for the Apo e...

D D Leonidas - One of the best experts on this subject based on the ideXlab platform.

  • inhibitor design for RibonucleAse A the binding of two 5 phosphAte uridine AnAlogues
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2009
    Co-Authors: V G Tsirkone, Kyriaki Dossi, C E Drakou, S E Zographos, Maria Kontou, D D Leonidas
    Abstract:

    In the quest for the rAtionAl design of selective And potent inhibitors for members of the pAncreAtic RibonucleAse A (RNAse A) fAmily of biomedicAl interest, the binding of uridine 5′-phosphAte (U5P) And uridine 5′-diphosphAte (UDP) to RNAse A hAve been investigAted using kinetic studies And X-rAy crystAllogrAphy. Both nucleotides Are competitive inhibitors of the enzyme, with K i vAlues of 4.0 And 0.65 mM, respectively. They bind to the Active site of the enzyme by Anchoring two molecules connected to eAch other by hydrogen bonds And vAn der WAAls interActions. While the first of the inhibitor molecules binds with its nucleobAse in the pyrimidinyl-binding subsite, the second is bound At the purine-preferring subsite. The unexpected binding of A pyrimidine At the purine-binding subsite hAs Added new importAnt elements to the rAtionAl design ApproAch for the discovery of new potent inhibitors of the RNAse A superfAmily.

  • towArd rAtionAl design of RibonucleAse inhibitors high resolution crystAl structure of A RibonucleAse A complex with A potent 3 5 pyrophosphAte linked dinucleotide inhibitor
    Biochemistry, 1999
    Co-Authors: D D Leonidas, Nello Russo, Robert Shapiro, L I Irons, K R Acharya
    Abstract:

    The crystAl structure of RibonucleAse A (RNAse A) in complex with pdUppA-3‘-p [5‘-phospho-2‘-deoxyuridine-3‘-pyrophosphAte (P‘→5‘) Adenosine 3‘-phosphAte] hAs been determined At 1.7 A resolution. This dinucleotide is the most potent low moleculAr weight inhibitor of RNAse A reported to dAte (Ki = 27 nM) And is Also effective AgAinst two mAjor nonpAncreAtic RNAses:  eosinophil-derived neurotoxin And RNAse-4; in All cAses, tight binding in lArge pArt derives from the unusuAl 3‘,5‘-pyrophosphAte internucleotide linkAge [Russo, N., And ShApiro, R. (1999) J. Biol. Chem. 274, 14902−14908]. The design of pdUppA-3‘-p wAs bAsed on the crystAl structure of RNAse A complexed with 5‘-diphosphoAdenosine 3‘-phosphAte (ppA-3‘-p) [LeonidAs, D. D., ShApiro, R., Irons, L. I., Russo, N., And AchAryA, K. R. (1997) Biochemistry 36, 5578−5588]. The Adenosine of pdUppA-3‘-p Adopts An AtypicAl syn conformAtion not observed for stAndArd Adenosine nucleotides bound to RNAse A. This conformAtion, which Allows extensive interActions...

P. J. Hore - One of the best experts on this subject based on the ideXlab platform.

  • Refolding of RibonucleAse A monitored by reAl-time photo-CIDNP NMR spectroscopy
    Journal of Biomolecular NMR, 2009
    Co-Authors: Kiminori Maeda, Howard J. Paisley, P. J. Hore
    Abstract:

    Photo-CIDNP NMR spectroscopy is A powerful method for investigAting the solvent Accessibility of histidine, tyrosine And tryptophAn residues in A protein. When coupled to reAl-time NMR, this technique Allows chAnges in the environments of these residues to be used As A probe of protein folding. In this pAper we describe experiments performed to monitor the refolding of RibonucleAse A following dilution from A high concentrAtion of chemicAl denAturAnt. These experiments provide A good exAmple of the utility of this technique which provides informAtion thAt is difficult to obtAin by other biophysicAl methods. ReAl-time photo-CIDNP meAsurements yield residue-specific kinetic dAtA pertAining to the folding reAction, interpreted in terms of current knowledge of the folding of bovine pAncreAtic RibonucleAse A.

Yoshiyuki Amemiya - One of the best experts on this subject based on the ideXlab platform.

  • The compActness of RibonucleAse A And reduced RibonucleAse A
    FEBS letters, 1998
    Co-Authors: Jun Mei Zhou, Ying-xin Fan, Hiroshi Kihara, Kazumoto Kimura, Yoshiyuki Amemiya
    Abstract:

    AbstrAct The compActness of RibonucleAse A with intAct disulfide bonds And reduced RibonucleAse A wAs investigAted by synchrotron smAll-Angle X-rAy scAttering. The R g vAlues And the KrAtky plots showed thAt non-reduced RibonucleAse A mAintAin A compAct shApe with A R g vAlue of About 17.3 A in 8 M ureA. The reduced RibonucleAse A is more expAnded, its R g vAlue is About 20 A in 50 mM Tris-HCl buffer At pH 8.1 contAining 20 mM DTT. Further expAnsions of reduced RibonucleAse A were observed in the presence of high concentrAtions of denAturAnts, indicAting thAt reduced RibonucleAse A is more expAnded And is in neither A rAndom coil [A. Noppert et Al., FEBS Lett. 380 (1996) 179–182] nor A compAct denAtured stAte [T.R. Sosnick And J. TrewhellA, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep RibonucleAse A in A compAct stAte in the presence of high concentrAtions of ureA.