Structure and function of a mutant of the cyclic AMP receptor protein: CRP*598.
Item
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Title
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Structure and function of a mutant of the cyclic AMP receptor protein: CRP*598.
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Identifier
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AAI9119672
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identifier
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9119672
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Creator
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Ren, Yun-ling.
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Contributor
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Adviser: Joseph S. Krakow
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Date
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1991
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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The cAMP receptor protein (CRP) acts to modulate the expression of a large number of E. coli genes. Binding of cAMP involves a conformational change in CRP with a resultant increase in the affinity and specificity for specific promoter-associated sites; unliganded CRP binds nonspecifically to DNA. CRP is composed of two identical subunits. The CRP monomer has a two-domain structure in which the large N-terminal domain is responsible for cAMP binding and subunit-subunit interaction and the smaller C-terminal domain is involved in DNA binding. One CRP mutant, CRP{dollar}\sp{lcub}\*{rcub}{dollar}598(Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. In the absence of cAMP, CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 shows a more open conformation than CRP, as indicated by its sensitivity to proteolytic attack and by 5,5{dollar}\sp\prime{dollar}-dithiobis(2-nitrobenzoic acid)-mediated subunit crosslinking. CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 can activate lac P{dollar}\sp{lcub}+{rcub}{dollar}-directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection indicates that cAMP-CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 and cGMP-CRP{dollar}\sp{lcub}\*{rcub}{dollar} form a stable complex with the ({dollar}\sp{lcub}32{rcub}{dollar}P) lac P{dollar}\sp{lcub}+{rcub}{dollar} fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation or transcription. The binding of cAMP and cGMP to CRP and CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 have been determined. The results indicate that the affinity of CRP and CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 for cGMP is relatively unchanged while the affinity of CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 for cAMP is approximately twenty times greater than that shown by CRP. Binding of cAMP by CRP and cGMP by CRP or CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 exhibits slight negative cooperativity. The major difference seen is that CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 binds cAMP with strong positive cooperativity. The positive cooperativity observed for binding of cAMP by CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 may be a consequence of effects modulated by altered subunit contacts and/or interdomain contacts. Three anti-CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 mAbs have been characterized. The dissociation constants for anti-CRP and anti-CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 mAbs to CRP and CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 are determined in the presence and absence of cyclic nucleotides. The results indicate that N{dollar}\sp6{dollar}-butyryl-cAMP induces conformational change of CRP/CRP{dollar}\sp{lcub}\*{rcub}{dollar}598 different from cAMP.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
