Abstract:
:The hinge domain encompasses amino acids 51-60 of lactose repressor (LacI) and plays an important role in its regulatory interaction with operator DNA. This segment makes both hinge-DNA and hinge-hinge' contacts that are critical to DNA binding. Furthermore, this small region serves as a central element in communicating the allosteric response to inducer. Introducing a disulfide bond between partner hinges within a dimer via the mutation V52C results in a protein that has increased affinity for O(1) operator DNA compared to wild-type LacI and abolishes allosteric response to inducer [Falcon, C. M., Swint-Kruse, L., and Matthews, K. S. (1997) J. Biol. Chem. 272, 26818]. We have established that this high affinity is maintained for the disulfide-linked protein even when symmetry and half-site spacing within the operator region are altered, whereas binding by the reduced protein, as for wild-type LacI, is severely diminished by these alterations. Interestingly, the allosteric response to inducer for V52C-oxidized remains intact for a small group of operator variants. Temperature studies demonstrate that the presence of the disulfide alters the thermodynamics of the protein-DNA interaction, with a DeltaC(p) of significantly smaller magnitude compared to wild-type LacI. The results presented here establish the hinge region as an important element not only for LacI high-affinity operator binding but also for the essential communication between ligand binding domains. Moreover, the results confirm that DNA sequence/conformation can profoundly influence allostery for this prototypic regulatory protein.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Falcon CM,Matthews KSdoi
10.1021/bi0114067subject
Has Abstractpub_date
2001-12-25 00:00:00pages
15650-9issue
51eissn
0006-2960issn
1520-4995pii
bi0114067journal_volume
40pub_type
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