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Perturbation from a distance: mutations that alter LacI function through long-range effects.
- Source :
-
Biochemistry [Biochemistry] 2003 Dec 02; Vol. 42 (47), pp. 14004-16. - Publication Year :
- 2003
-
Abstract
- Allosteric modification of ligand binding is central to LacI transcription control. Recently, the conformational change between LacI operator- and inducer-bound states was simulated with targeted molecular dynamics (TMD) [Flynn, T. C., Swint-Kruse, L., Kong, Y., Booth, C., Matthews, K. S., and Ma, J. (2003) Protein Sci., 12, 2523-2541]. Atomic-level analyses of TMD results indicate the structural importance of the core pivot region that connects the N- and C-subdomains flanking the inducer-binding site. Further, a number of LacI mutations in the core pivot have been identified recently by their altered behaviors in phenotypic screens. Biochemical characterization of three of these variants-L148F, S151P, and P320A-provides an opportunity to directly explore the role of the core pivot in repressor function. For L148F, inducer IPTG binding affinity is strengthened, whereas O(1) operator DNA binding is diminished approximately 30-fold. In contrast, O(1) binding is increased for S151P, whereas IPTG binding is decreased. UV-difference spectroscopy and urea denaturation indicate long-range effects in both variants. Interestingly, P320A binds to DNA approximately 4-fold more tightly than wild-type, yet inducer binding is unaffected. To examine linkage between the core pivot and DNA binding domains, the L148F substitution was combined with Q60G, a previously known mutant with enhanced operator affinity. The double mutant exhibits the properties of both parent proteins, resulting in near wild-type DNA binding affinity and enhanced inducer sensitivity. These features may render Q60G/L148F more cost-effective in technological applications than wild-type repressor. As a group, the behaviors of the core pivot mutants are consistent with the allosteric structural role predicted for this region by TMD and reflect the significant long-range impact that single substitutions can elicit on protein function.
- Subjects :
- Amino Acid Substitution genetics
Bacterial Proteins isolation & purification
DNA-Binding Proteins chemistry
DNA-Binding Proteins genetics
Escherichia coli Proteins chemistry
Escherichia coli Proteins genetics
Isopropyl Thiogalactoside chemistry
Lac Repressors
Lactose chemistry
Melibiose chemistry
Operator Regions, Genetic
Phenotype
Protein Conformation
Protein Denaturation
Protein Structure, Tertiary genetics
Repressor Proteins isolation & purification
Spectrometry, Fluorescence
Spectrophotometry, Ultraviolet
Urea chemistry
Bacterial Proteins chemistry
Bacterial Proteins genetics
Mutagenesis, Site-Directed
Repressor Proteins chemistry
Repressor Proteins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 42
- Issue :
- 47
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 14636069
- Full Text :
- https://doi.org/10.1021/bi035116x