Abstract:
:The allosteric activation of the T127-->L mutant of 3',5'-cyclic adenosine monophosphate (cAMP) receptor protein (CRP) by cAMP changes from an exothermic, independent two-site binding mechanism at pH 7.0 to an endothermic, interacting two-site binding mechanism at pH 5.2, similar to that observed for CRP at pH 7.0 and 5.2. Since the T127-->L mutation at the subunit interface of the CRP dimer creates a more perfect leucine-zipper motif, it is believed to increase the intersubunit association and the stability of the CRP, as is observed by the higher thermal stability of the T127L mutant relative to that of CRP in differential scanning calorimetry (DSC) measurements. The DSC scans also exhibit a single thermal denaturation transition for CRP and a S128A mutant from pH 5.2 to 7. 0, while the broader transition peak of the T127L mutant becomes resolvable into two transitions below pH < or =5.2. Circular dichroism measurements on T127L and CRP at pH 7.0 and 5.2 show changes in the tertiary structure of both proteins with the exception of the tertiary structure around the two tryptophan residues in the amino-terminal domain. Although gel electrophoresis of the proteolysis (pH 5.2) products of T127L, CRP, and their cAMP- and cGMP-ligated complexes shows the subunit band and an amino-terminal domain fragment band, the fully allosterically activated complexes of T127L and CRP show the amino-terminal domain fragment band but not the subunit band. The results are interpreted in terms of the allosteric activation of CRP by cAMP by a conformational change from an "open" to a "closed" form of CRP, which involves changes in the tertiary structure of the carboxyl-terminal domains that are partially induced by an increase in the intersubunit association in T127L. While T127L retains its intersubunit association from pH 5.2 to 7.0, changes occur in the carboxyl-terminal domain so that the endothermic, allosteric activation mechanism of CRP by cAMP is restored in T127L at pH 5.2.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Shi Y,Wang S,Schwarz FPdoi
10.1021/bi000225msubject
Has Abstractpub_date
2000-06-20 00:00:00pages
7300-8issue
24eissn
0006-2960issn
1520-4995pii
bi000225mjournal_volume
39pub_type
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