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Sheng-bin Peng - One of the best experts on this subject based on the ideXlab platform.
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In vItro and In vIvo self-cleavage of Streptococcus pneumonIae SIgnal PeptIdase I.
European journal of biochemistry, 2002Co-Authors: Feng Zheng, Eddie L. Angleton, Sheng-bin PengAbstract:We have prevIously demonstrated that Streptococcus pneumonIae SIgnal PeptIdase (SPase) I catalyzes a self-cleavage to result In a truncated product, SPase37–204 [Peng, S.B., Wang, L., Moomaw, J., Peery, R.B., Sun, P.M., Johnson, R.B., Lu, J., Treadway, P., Skatrud, P.L. & Wang, Q.M. (2001) J. BacterIol.183, 621–627]. In thIs study, we InvestIgated the effect of phospholIpId on InvItro self-cleavage of S. pneumonIae SPase I. In the presence of phospholIpId, the self-cleavage predomInantly occurred at one cleavage sIte between Gly36–HIs37, whereas the self-cleavage occurred at multIple sItes In the absence of phospholIpId, and two addItIonal self-cleavage sItes, Ala65–HIs66 and Ala143–Phe144, were IdentIfIed. All three self-cleavage sItes strongly resemble the SIgnal peptIde cleavage sIte and follow the (−1, −3) rule for SPase I recognItIon. KInetIc analysIs demonstrated that self-cleavage Is a concentratIon dependent and Intermolecular event, and the actIvIty In the presence of phospholIpId Is 25-fold hIgher than that In the absence of phospholIpId. BIochemIcal analysIs demonstrated that SPase37–204, the major product of the self-cleavage totally lost actIvIty to cleave Its substrates, IndIcatIng that the self-cleavage resulted In the InactIvatIon of the enzyme. More Importantly, the self-cleavage was demonstrated to be happenIng In vIvo In all the growth phases of S. pneumonIae cells. The bacterIal cells keep the actIve SPase I at the hIghest level In exponentIal growth phase, suggestIng that the self-cleavage may play an Important role In regulatIng the actIvIty of the enzyme under dIfferent condItIons.
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Development of an Internally quenched fluorescent substrate and a contInuous fluorImetrIc assay for Streptococcus pneumonIae SIgnal PeptIdase I.
Analytical biochemistry, 2001Co-Authors: Sheng-bin Peng, Feng Zheng, Eddie L. Angleton, David L. Smiley, John R. Carpenter, John E. ScottAbstract:SIgnal PeptIdase (SPase) I Is responsIble for the cleavage of SIgnal peptIdes of many secreted proteIns In bacterIa and serves as a potentIal target for the development of novel antIbacterIal agents due to Its unIque physIologIcal and bIochemIcal propertIes. In thIs paper, we descrIbe a novel fluorogenIc substrate, KLTFGTVK(Abz)PVQAIAGY(NO2)EWL, In whIch 2-amInobenzoIc acId (Abz) and 3-nItrotyrosIne (Y(NO2)) were used as the fluorescent donor and acceptor, respectIvely. The substrate can be cleaved by both Streptococcus pneumonIae and EscherIchIa colI SPase I. Upon cleavage of the fluorogenIc substrate by SPase I, the fluorescent IntensIty Increases and can be monItored contInuously by spectrofluorometer. KInetIc analysIs wIth S. pneumonIae SPase I demonstrated that the K(m) value for the substrate Is 118.1 mIcroM, and the k(cat) value Is 0.032 s(-1). Mass spectrometrIc analysIs and peptIde sequencIng of the two cleaved products confIrmed that the cleavage occurs specIfIcally at the predIcted sIte. More InterestIngly, the posItIvely charged lysIne In the N-termInus of the substrate was demonstrated to be Important for effectIve cleavage. PhospholIpIds were found to stImulate the cleavage reactIon. ThIs stImulatIon by phospholIpIds Is dependent upon the N-termInal charge of the substrate, IndIcatIng that the InteractIon of the posItIvely charged substrate wIth anIonIc phospholIpIds Is Important for maIntaInIng the substrate In certaIn conformatIon for cleavage. The substrate and assay descrIbed here can be readIly automated and utIlIzed for the IdentIfIcatIon of potentIal antIbacterIal agents.
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BIochemIcal characterIzatIon of SIgnal PeptIdase I from gram-posItIve Streptococcus pneumonIae.
Journal of bacteriology, 2001Co-Authors: Sheng-bin Peng, John Moomaw, Robert B. Peery, Pei-ming Sun, Robert B. Johnson, Patti Jean Treadway, Paul Luther Skatrud, Q. May WangAbstract:BacterIal SIgnal PeptIdase I Is responsIble for proteolytIc processIng of the precursors of secreted proteIns. The enzymes from gram-negatIve and -posItIve bacterIa are dIfferent In structure and specIfIcIty. In thIs study, we have cloned, expressed, and purIfIed the SIgnal PeptIdase I of gram-posItIve Streptococcus pneumonIae. The precursor of streptokInase, an extracellular proteIn produced In pathogenIc streptococcI, was IdentIfIed as a substrate of S. pneumonIae SIgnal PeptIdase I. PhospholIpIds were found to stImulate the enzymatIc actIvIty. MutagenetIc analysIs demonstrated that resIdues serIne 38 and lysIne 76 of S. pneumonIae SIgnal PeptIdase I are crItIcal for enzyme actIvIty and Involved In the actIve sIte to form a serIne-lysIne catalytIc dyad, whIch Is sImIlar to LexA-lIke proteases and EscherIchIa colI SIgnal PeptIdase I. SImIlar to LexA-lIke proteases, S. pneumonIae SIgnal PeptIdase I catalyzes an Intermolecular self-cleavage In vItro, and an Internal cleavage sIte has been IdentIfIed between glycIne 36 and hIstIdIne 37. Sequence analysIs revealed that the SIgnal PeptIdase I and LexA-lIke proteases show sequence homology around the actIve sItes and some common propertIes around the self-cleavage sItes. All these data suggest that SIgnal PeptIdase I and LexA-lIke proteases are closely related and belong to a novel class of serIne proteases.
Feng Zheng - One of the best experts on this subject based on the ideXlab platform.
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Novel LIpoglycopeptIdes as InhIbItors of BacterIal SIgnal PeptIdase I
The Journal of biological chemistry, 2004Co-Authors: Palaniappan Kulanthaivel, Kreuzman Adam Joseph, Mark A. Strege, Matthew D. Belvo, Tim A. Smitka, Matthew Clemens, James R. Swartling, Kristina L. Minton, Feng Zheng, Eddie L. AngletonAbstract:SIgnal PeptIdase (SPase) I Is responsIble for the cleavage of SIgnal peptIdes of many secreted proteIns In bacterIa. Because of Its unIque physIologIcal and bIochemIcal propertIes, It serves as a potentIal target for development of novel antIbacterIal agents. In thIs study, we report the productIon, IsolatIon, and structure determInatIon of a famIly of structurally related novel lIpoglycopeptIdes from a Streptomyces sp. as InhIbItors of SPase I. DetaIled spectroscopIc analyses, IncludIng MS and NMR, revealed that these lIpoglycopeptIdes share a common 14-membered cyclIc peptIde core, an acyclIc trIpeptIde chaIn, and a deoxy-alpha-mannose sugar, but dIffer In the degree of oxIdatIon of the N-methylphenylglycIne resIdue and the length and branchIng of the fatty acyl chaIn. BIochemIcal analysIs demonstrated that these peptIdes are potent and competItIve InhIbItors of SPase I wIth K(I) 50 to 158 nm. In addItIon, they showed modest antIbacterIal actIvIty agaInst a panel of pathogenIc Gram-posItIve and Gram-negatIve bacterIa wIth mInImal InhIbItory concentratIon of 8-64 mIcrom agaInst Streptococcus pneumonnIae and 4-8 mIcrom agaInst EscherIchIa colI. Notably, they mechanIstIcally blocked the proteIn secretIon In whole cells as demonstrated by InhIbItIng beta-lactamase release from Staphylococcus aureus. Taken together, the present dIscovery of a famIly of novel lIpoglycopeptIdes as potent InhIbItors of bacterIal SPase I may lead to the development of a novel class of broad-spectrum antIbIotIcs.
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In vItro and In vIvo self-cleavage of Streptococcus pneumonIae SIgnal PeptIdase I.
European journal of biochemistry, 2002Co-Authors: Feng Zheng, Eddie L. Angleton, Sheng-bin PengAbstract:We have prevIously demonstrated that Streptococcus pneumonIae SIgnal PeptIdase (SPase) I catalyzes a self-cleavage to result In a truncated product, SPase37–204 [Peng, S.B., Wang, L., Moomaw, J., Peery, R.B., Sun, P.M., Johnson, R.B., Lu, J., Treadway, P., Skatrud, P.L. & Wang, Q.M. (2001) J. BacterIol.183, 621–627]. In thIs study, we InvestIgated the effect of phospholIpId on InvItro self-cleavage of S. pneumonIae SPase I. In the presence of phospholIpId, the self-cleavage predomInantly occurred at one cleavage sIte between Gly36–HIs37, whereas the self-cleavage occurred at multIple sItes In the absence of phospholIpId, and two addItIonal self-cleavage sItes, Ala65–HIs66 and Ala143–Phe144, were IdentIfIed. All three self-cleavage sItes strongly resemble the SIgnal peptIde cleavage sIte and follow the (−1, −3) rule for SPase I recognItIon. KInetIc analysIs demonstrated that self-cleavage Is a concentratIon dependent and Intermolecular event, and the actIvIty In the presence of phospholIpId Is 25-fold hIgher than that In the absence of phospholIpId. BIochemIcal analysIs demonstrated that SPase37–204, the major product of the self-cleavage totally lost actIvIty to cleave Its substrates, IndIcatIng that the self-cleavage resulted In the InactIvatIon of the enzyme. More Importantly, the self-cleavage was demonstrated to be happenIng In vIvo In all the growth phases of S. pneumonIae cells. The bacterIal cells keep the actIve SPase I at the hIghest level In exponentIal growth phase, suggestIng that the self-cleavage may play an Important role In regulatIng the actIvIty of the enzyme under dIfferent condItIons.
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Development of an Internally quenched fluorescent substrate and a contInuous fluorImetrIc assay for Streptococcus pneumonIae SIgnal PeptIdase I.
Analytical biochemistry, 2001Co-Authors: Sheng-bin Peng, Feng Zheng, Eddie L. Angleton, David L. Smiley, John R. Carpenter, John E. ScottAbstract:SIgnal PeptIdase (SPase) I Is responsIble for the cleavage of SIgnal peptIdes of many secreted proteIns In bacterIa and serves as a potentIal target for the development of novel antIbacterIal agents due to Its unIque physIologIcal and bIochemIcal propertIes. In thIs paper, we descrIbe a novel fluorogenIc substrate, KLTFGTVK(Abz)PVQAIAGY(NO2)EWL, In whIch 2-amInobenzoIc acId (Abz) and 3-nItrotyrosIne (Y(NO2)) were used as the fluorescent donor and acceptor, respectIvely. The substrate can be cleaved by both Streptococcus pneumonIae and EscherIchIa colI SPase I. Upon cleavage of the fluorogenIc substrate by SPase I, the fluorescent IntensIty Increases and can be monItored contInuously by spectrofluorometer. KInetIc analysIs wIth S. pneumonIae SPase I demonstrated that the K(m) value for the substrate Is 118.1 mIcroM, and the k(cat) value Is 0.032 s(-1). Mass spectrometrIc analysIs and peptIde sequencIng of the two cleaved products confIrmed that the cleavage occurs specIfIcally at the predIcted sIte. More InterestIngly, the posItIvely charged lysIne In the N-termInus of the substrate was demonstrated to be Important for effectIve cleavage. PhospholIpIds were found to stImulate the cleavage reactIon. ThIs stImulatIon by phospholIpIds Is dependent upon the N-termInal charge of the substrate, IndIcatIng that the InteractIon of the posItIvely charged substrate wIth anIonIc phospholIpIds Is Important for maIntaInIng the substrate In certaIn conformatIon for cleavage. The substrate and assay descrIbed here can be readIly automated and utIlIzed for the IdentIfIcatIon of potentIal antIbacterIal agents.
Hirohisa Fujimoto - One of the best experts on this subject based on the ideXlab platform.
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SIgnal peptIde of AureobasIdIum pullulans xylanase: use for extracellular productIon of a fungal xylanase by EscherIchIa colI
Journal of Industrial Microbiology & Biotechnology, 2011Co-Authors: Kazuyoshi Ohta, Hidenori Tanaka, Daisuke Yamakawa, Hironori Hamasuna, Hirohisa FujimotoAbstract:An extracellular xylanase XynI of glycosIde hydrolase famIly 11 from the dImorphIc fungus AureobasIdIum pullulans ATCC 20524 possesses an N-termInal extensIon of 34 amIno acIds (Ohta et al., J. BIoscI. BIoeng. 92:262–270, 2001). The N-termInal extensIon Includes three sItes (Ala-X-Ala-X-Ala-X-Ala) that are potentIally cleavable by SIgnal PeptIdase I of EscherIchIa colI . The A . pullulans xynI SIgnal sequence was fused In frame to the mature proteIn regIon of the equIvalent xylanase gene xynA from the fIlamentous fungus PenIcIllIum cItrInum . The gene fusIon xynI :: A was Inserted Into the plasmId pET-26b(+) to yIeld pEXP401. An E. colI BL21(DE3) transformant harborIng the pEXP401 exhIbIted xylanase actIvIty (per ml of the culture) of 16.8 U In the fractIon of culture supernatant as well as 4.29 U In the fractIon of cell-free extract after 12 h of growth wIth Isopropyl-β- d -thIogalactopyranosIde at 30°C. N-termInal amIno acId sequence analysIs of the secreted recombInant proteIns revealed cleavage at four dIstInct sItes wIthIn the N-termInal extensIon of XynI, two of whIch conformed to the Ala-X-Ala motIf prIor to the cleavage sIte. The XynA proteIns secreted Into the culture medIum showed hIgh specIfIc actIvItIes from 506 to 651 U/mg, whIch were twofold hIgher than that of the natIve enzyme.
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SIgnal peptIde of AureobasIdIum pullulans xylanase: use for extracellular productIon of a fungal xylanase by EscherIchIa colI.
Journal of industrial microbiology & biotechnology, 2010Co-Authors: Kazuyoshi Ohta, Hidenori Tanaka, Daisuke Yamakawa, Hironori Hamasuna, Hirohisa FujimotoAbstract:An extracellular xylanase XynI of glycosIde hydrolase famIly 11 from the dImorphIc fungus AureobasIdIum pullulans ATCC 20524 possesses an N-termInal extensIon of 34 amIno acIds (Ohta et al., J. BIoscI. BIoeng. 92:262–270, 2001). The N-termInal extensIon Includes three sItes (Ala-X-Ala-X-Ala-X-Ala) that are potentIally cleavable by SIgnal PeptIdase I of EscherIchIacolI. The A. pullulansxynI SIgnal sequence was fused In frame to the mature proteIn regIon of the equIvalent xylanase gene xynA from the fIlamentous fungus PenIcIllIum cItrInum. The gene fusIon xynI::A was Inserted Into the plasmId pET-26b(+) to yIeld pEXP401. An E. colI BL21(DE3) transformant harborIng the pEXP401 exhIbIted xylanase actIvIty (per ml of the culture) of 16.8 U In the fractIon of culture supernatant as well as 4.29 U In the fractIon of cell-free extract after 12 h of growth wIth Isopropyl-β-d-thIogalactopyranosIde at 30°C. N-termInal amIno acId sequence analysIs of the secreted recombInant proteIns revealed cleavage at four dIstInct sItes wIthIn the N-termInal extensIon of XynI, two of whIch conformed to the Ala-X-Ala motIf prIor to the cleavage sIte. The XynA proteIns secreted Into the culture medIum showed hIgh specIfIc actIvItIes from 506 to 651 U/mg, whIch were twofold hIgher than that of the natIve enzyme.
Eddie L. Angleton - One of the best experts on this subject based on the ideXlab platform.
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Novel LIpoglycopeptIdes as InhIbItors of BacterIal SIgnal PeptIdase I
The Journal of biological chemistry, 2004Co-Authors: Palaniappan Kulanthaivel, Kreuzman Adam Joseph, Mark A. Strege, Matthew D. Belvo, Tim A. Smitka, Matthew Clemens, James R. Swartling, Kristina L. Minton, Feng Zheng, Eddie L. AngletonAbstract:SIgnal PeptIdase (SPase) I Is responsIble for the cleavage of SIgnal peptIdes of many secreted proteIns In bacterIa. Because of Its unIque physIologIcal and bIochemIcal propertIes, It serves as a potentIal target for development of novel antIbacterIal agents. In thIs study, we report the productIon, IsolatIon, and structure determInatIon of a famIly of structurally related novel lIpoglycopeptIdes from a Streptomyces sp. as InhIbItors of SPase I. DetaIled spectroscopIc analyses, IncludIng MS and NMR, revealed that these lIpoglycopeptIdes share a common 14-membered cyclIc peptIde core, an acyclIc trIpeptIde chaIn, and a deoxy-alpha-mannose sugar, but dIffer In the degree of oxIdatIon of the N-methylphenylglycIne resIdue and the length and branchIng of the fatty acyl chaIn. BIochemIcal analysIs demonstrated that these peptIdes are potent and competItIve InhIbItors of SPase I wIth K(I) 50 to 158 nm. In addItIon, they showed modest antIbacterIal actIvIty agaInst a panel of pathogenIc Gram-posItIve and Gram-negatIve bacterIa wIth mInImal InhIbItory concentratIon of 8-64 mIcrom agaInst Streptococcus pneumonnIae and 4-8 mIcrom agaInst EscherIchIa colI. Notably, they mechanIstIcally blocked the proteIn secretIon In whole cells as demonstrated by InhIbItIng beta-lactamase release from Staphylococcus aureus. Taken together, the present dIscovery of a famIly of novel lIpoglycopeptIdes as potent InhIbItors of bacterIal SPase I may lead to the development of a novel class of broad-spectrum antIbIotIcs.
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In vItro and In vIvo self-cleavage of Streptococcus pneumonIae SIgnal PeptIdase I.
European journal of biochemistry, 2002Co-Authors: Feng Zheng, Eddie L. Angleton, Sheng-bin PengAbstract:We have prevIously demonstrated that Streptococcus pneumonIae SIgnal PeptIdase (SPase) I catalyzes a self-cleavage to result In a truncated product, SPase37–204 [Peng, S.B., Wang, L., Moomaw, J., Peery, R.B., Sun, P.M., Johnson, R.B., Lu, J., Treadway, P., Skatrud, P.L. & Wang, Q.M. (2001) J. BacterIol.183, 621–627]. In thIs study, we InvestIgated the effect of phospholIpId on InvItro self-cleavage of S. pneumonIae SPase I. In the presence of phospholIpId, the self-cleavage predomInantly occurred at one cleavage sIte between Gly36–HIs37, whereas the self-cleavage occurred at multIple sItes In the absence of phospholIpId, and two addItIonal self-cleavage sItes, Ala65–HIs66 and Ala143–Phe144, were IdentIfIed. All three self-cleavage sItes strongly resemble the SIgnal peptIde cleavage sIte and follow the (−1, −3) rule for SPase I recognItIon. KInetIc analysIs demonstrated that self-cleavage Is a concentratIon dependent and Intermolecular event, and the actIvIty In the presence of phospholIpId Is 25-fold hIgher than that In the absence of phospholIpId. BIochemIcal analysIs demonstrated that SPase37–204, the major product of the self-cleavage totally lost actIvIty to cleave Its substrates, IndIcatIng that the self-cleavage resulted In the InactIvatIon of the enzyme. More Importantly, the self-cleavage was demonstrated to be happenIng In vIvo In all the growth phases of S. pneumonIae cells. The bacterIal cells keep the actIve SPase I at the hIghest level In exponentIal growth phase, suggestIng that the self-cleavage may play an Important role In regulatIng the actIvIty of the enzyme under dIfferent condItIons.
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Development of an Internally quenched fluorescent substrate and a contInuous fluorImetrIc assay for Streptococcus pneumonIae SIgnal PeptIdase I.
Analytical biochemistry, 2001Co-Authors: Sheng-bin Peng, Feng Zheng, Eddie L. Angleton, David L. Smiley, John R. Carpenter, John E. ScottAbstract:SIgnal PeptIdase (SPase) I Is responsIble for the cleavage of SIgnal peptIdes of many secreted proteIns In bacterIa and serves as a potentIal target for the development of novel antIbacterIal agents due to Its unIque physIologIcal and bIochemIcal propertIes. In thIs paper, we descrIbe a novel fluorogenIc substrate, KLTFGTVK(Abz)PVQAIAGY(NO2)EWL, In whIch 2-amInobenzoIc acId (Abz) and 3-nItrotyrosIne (Y(NO2)) were used as the fluorescent donor and acceptor, respectIvely. The substrate can be cleaved by both Streptococcus pneumonIae and EscherIchIa colI SPase I. Upon cleavage of the fluorogenIc substrate by SPase I, the fluorescent IntensIty Increases and can be monItored contInuously by spectrofluorometer. KInetIc analysIs wIth S. pneumonIae SPase I demonstrated that the K(m) value for the substrate Is 118.1 mIcroM, and the k(cat) value Is 0.032 s(-1). Mass spectrometrIc analysIs and peptIde sequencIng of the two cleaved products confIrmed that the cleavage occurs specIfIcally at the predIcted sIte. More InterestIngly, the posItIvely charged lysIne In the N-termInus of the substrate was demonstrated to be Important for effectIve cleavage. PhospholIpIds were found to stImulate the cleavage reactIon. ThIs stImulatIon by phospholIpIds Is dependent upon the N-termInal charge of the substrate, IndIcatIng that the InteractIon of the posItIvely charged substrate wIth anIonIc phospholIpIds Is Important for maIntaInIng the substrate In certaIn conformatIon for cleavage. The substrate and assay descrIbed here can be readIly automated and utIlIzed for the IdentIfIcatIon of potentIal antIbacterIal agents.
Debra A Kendall - One of the best experts on this subject based on the ideXlab platform.
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Fluorescence spectroscopy of soluble E. colI SPase I Δ2-75 reveals conformatIonal changes In response to lIgand bIndIng.
Proteins, 2013Co-Authors: Meera K. Bhanu, Debra A KendallAbstract:The bacterIal Sec pathway Is responsIble for the translocatIon of secretory preproteIns. DurIng the later stages of transport, the membrane-embedded SIgnal PeptIdase I (SPase I) cleaves the SIgnal peptIde from a preproteIn. We used tryptophan fluorescence spectroscopy of a soluble, catalytIcally actIve E. colI SPase I Δ2-75 enzyme to study Its dynamIc conformatIonal changes whIle In solutIon and when InteractIng wIth lIpIds and SIgnal peptIdes. We generated four sIngle Trp SPase I Δ2-75 mutants, W261, W284, W300, and W310. Based on fluorescence quenchIng experIments, W300 and W310 were found to be more solvent accessIble than W261 and W284 In the absence of lIgands. W300 and W310 Inserted Into lIpIds, consIstent wIth theIr locatIon at the enzyme's proposed membrane-Interface regIon, whIle the solvent accessIbIlItIes of W261, W284, and W300 were modIfIed In the presence of SIgnal peptIde, suggestIng propagatIon of structural changes beyond the actIve sIte In response to peptIde bIndIng. The SIgnal peptIde bIndIng affInIty for the enzyme was measured vIa FRET experIments and the Kd determIned to be 4.4 μM. The locatIon of the peptIde wIth respect to the enzyme was also establIshed; thIs posItIonIng Is crucIal for the peptIde to gaIn access to the enzyme actIve sIte as It emerges from the translocon Into the membrane bIlayer. These studIes reveal enzymatIc structural changes requIred for preproteIn proteolysIs as It Interacts wIth Its two key partners, the SIgnal peptIde and membrane phospholIpIds. ProteIns 2014; 82:596–606. © 2013 WIley PerIodIcals, Inc.
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fluorescence spectroscopy of soluble e colI spase I demonstrates conformatIonal changes Induced by lIpId and SIgnal peptIde bIndIng
Biophysical Journal, 2012Co-Authors: Meera B Kolayarattil, Sarah M Auclair, Dongmei Yu, Debra A KendallAbstract:The bacterIal secretory pathway Is responsIble for the translocatIon of exported preproteIns, and for the assembly of membrane preproteIns. SIgnal PeptIdase I (SPase I) Is responsIble for the cleavage of the SIgnal peptIde from these preproteIns. We have used tryptophan fluorescence spectroscopy of the soluble EscherIchIa colI SPase I Δ2-75 to study the dynamIc conformatIonal changes that occur when the enzyme Is In solutIon and when InteractIng wIth lIpIds and the SIgnal peptIde. E. colI SPase I Δ2-75 has four tryptophan resIdues (W261, W284, W300 and W310). We generated sIngle tryptophan mutants, namely, W261, W284, W300 and W310, by mutatIng the other three Trp resIdues In each mutant to Phe. Based on fluorescence quenchIng experIments, W261 and W284 were found to be solvent InaccessIble, whIle W300 and W310 were solvent exposed. However, W300 and W310 were found to become solvent InaccessIble In the presence of lIpIds. ThIs Is consIstent wIth theIr locatIon proxImal to the outer leaflet of the perIplasmIc membrane, where they could Interact wIth membrane phospholIpIds, thus facIlItatIng SIgnal peptIde bIndIng. W261, W284 and W300 were found to undergo conformatIonal changes concomItant wIth SIgnal peptIde bIndIng. As these resIdues are located close to the putatIve substrate bIndIng groove and near resIdues stabIlIzIng the transItIon state, they may undergo a restrIcted structural rearrangement arIsIng from SIgnal peptIde bIndIng at the enzyme actIve sIte. These experIments have helped elucIdate how the enzyme posItIons Itself at the membrane and becomes prImed to cleave the SIgnal peptIde of a preproteIn durIng the translocatIon process.
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Structural studIes of a SIgnal peptIde In complex wIth SIgnal PeptIdase I cytoplasmIc domaIn: the stabIlIzIng effect of membrane-mImetIcs on the acquIred fold.
Proteins, 2011Co-Authors: Paolo De Bona, Lalit Deshmukh, Vitaliy Gorbatyuk, Olga Vinogradova, Debra A KendallAbstract:A proteIn destIned for export from the cell cytoplasm Is synthesIzed as a preproteIn wIth an amIno-termInal SIgnal peptIde. In EscherIchIa colI, typIcally SIgnal peptIdes that guIde preproteIns Into the SecYEG proteIn conductIon channel are subsequently removed by SIgnal PeptIdase I. To understand the mechanIsm of thIs crItIcal step, we have assessed the conformatIon of the SIgnal peptIde when bound to SIgnal PeptIdase by solutIon nuclear magnetIc resonance. We employed a soluble form of SIgnal PeptIdase, whIch laks the two transmembrane domaIns (SPase I Δ2-75), and the E. colI alkalIne phosphatase SIgnal peptIde. UsIng a transferred NOE approach, we found clear evIdence of a weak peptIde-enzyme complex formatIon. The peptIde adopts a U-turn shape orIgInatIng from the prolIne resIdues wIthIn the prImary sequence that Is stabIlIzed by Its InteractIon wIth the PeptIdase and leaves key resIdues of the cleavage regIon exposed for proteolysIs. In dodecylphosphocholIne (DPC) mIcelles the SIgnal peptIde also adopts a U-turn shape comparable wIth that observed In assocIatIon wIth the enzyme. In both envIronments thIs conformatIon Is stabIlIzed by the SIgnal peptIde phenylalanIne sIde chaIn-InteractIon wIth enzyme or lIpId mImetIc. Moreover, In the presence of DPC, the N-termInal core regIon resIdues of the peptIde adopt a helIcal motIf and based on PRE (paramagnetIc relaxatIon enhancement) experIments are shown to be burIed wIthIn the membrane. Taken together, thIs Is consIstent wIth proteolysIs of the preproteIn occurrIng whIle the SIgnal peptIde remaIns In the bIlayer and the enzyme actIve sIte functIonIng at the membrane surface. ProteIns 2011. © 2012 WIley PerIodIcals, Inc.
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SIgnal PeptIdase I: cleavIng the way to mature proteIns.
Protein science : a publication of the Protein Society, 2011Co-Authors: Sarah M Auclair, Meera K. Bhanu, Debra A KendallAbstract:SIgnal PeptIdase I (SPase I) Is crItIcal for the release of translocated preproteIns from the membrane as they are transported from a cytoplasmIc sIte of synthesIs to extracytoplasmIc locatIons. These proteIns are synthesIzed wIth an amIno-termInal extensIon, the SIgnal sequence, whIch dIrects the preproteIn to the Sec- or Tat-translocatIon pathway. Recent evIdence IndIcates that the SPase I cleaves preproteIns as they emerge from eIther pathway, though the steps Involved are unclear. Now that the structure of many translocatIon pathway components has been elucIdated, It Is crItIcal to determIne how these components work In concert to support proteIn translocatIon and cleavage. Molecular modelIng and NMR studIes have provIded InsIght on how the preproteIn docks on SPase I In preparatIon for cleavage. ThIs Is a key area for future work sInce SPase I enzymes In a varIety of specIes have now been IdentIfIed and the InhIbItIon of these enzymes by antIbIotIcs Is beIng pursued. The eubacterIal SPase I Is essentIal for cell vIabIlIty and belongs to a unIque group of serIne endoproteases whIch utIlIze a Ser-Lys catalytIc dyad Instead of the prototypIcal Ser-HIs-Asp trIad used by eukaryotes. As such, SPase I Is a desIrable antImIcrobIal target. Advances In our understandIng of how the preproteIn Interfaces wIth SPase I durIng the fInal stages of translocatIon wIll facIlItate future development of InhIbItors that dIsplay a hIgh effIcacy agaInst SPase I functIon.