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Energetics of ligand-induced conformational flexibility in the lactose permease of Escherichia coli.

ABSTRACT: Isothermal titration calorimetry has been applied to characterize the thermodynamics of ligand binding to wild-type lactose permease (LacY) and a mutant (C154G) that strongly favors an inward facing conformation. The affinity of wild-type or mutant LacY for ligand and the change in free energy (DeltaG) upon binding are similar. However, with the wild type, the change in free energy upon binding is due primarily to an increase in the entropic free energy component (TDeltaS), whereas in marked contrast, an increase in enthalpy (DeltaH) is responsible for DeltaG in the mutant. Thus, wild-type LacY behaves as if there are multiple ligand-bound conformational states, whereas the mutant is severely restricted. The findings also indicate that the structure of the mutant represents a conformational intermediate in the overall transport cycle.

SUBMITTER: Nie Y

PROVIDER: S-EPMC2793331 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Energetics of ligand-induced conformational flexibility in the lactose permease of Escherichia coli.

Nie Yiling Y Smirnova Irina I Kasho Vladimir V Kaback H Ronald HR

The Journal of biological chemistry 20060926 47

Isothermal titration calorimetry has been applied to characterize the thermodynamics of ligand binding to wild-type lactose permease (LacY) and a mutant (C154G) that strongly favors an inward facing conformation. The affinity of wild-type or mutant LacY for ligand and the change in free energy (DeltaG) upon binding are similar. However, with the wild type, the change in free energy upon binding is due primarily to an increase in the entropic free energy component (TDeltaS), whereas in marked con ...[more]

PMID: 17003033

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Ligand-induced conformational changes and conformational dynamics in the solution structure of the lactose repressor protein.

Project description:We present here the results of a series of small-angle X-ray scattering studies aimed at understanding the role of conformational changes and structural flexibility in DNA binding and allosteric signaling in a bacterial transcription regulator, lactose repressor protein (LacI). Experiments were designed to detect possible conformational changes that occur when LacI binds either DNA or the inducer IPTG, or both. Our studies included the native LacI dimer of homodimers and a dimeric variant (R3), enabling us to probe conformational changes within the homodimers and distinguish them from those involving changes in the homodimer-homodimer relationships. The scattering data indicate that removal of operator DNA (oDNA) from R3 results in an unfolding and extension of the hinge helix that connects the LacI regulatory and DNA-binding domains. In contrast, only very subtle conformational changes occur in the R3 dimer-oDNA complex upon IPTG binding, indicative of small adjustments in the orientations of domains and/or subdomains within the structure. The binding of IPTG to native (tetrameric) LacI-oDNA complexes also appears to facilitate a modest change in the average homodimer-homodimer disposition. Notably, the crystal structure of the native LacI-oDNA complex differs significantly from the average solution conformation. The solution scattering data are best fit by an ensemble of structures that includes (1) approximately 60% of the V-shaped dimer of homodimers observed in the crystal structure and (2) approximately 40% of molecules with more "open" forms, such as those generated when the homodimers move with respect to each other about the tetramerization domain. In gene regulation, such a flexible LacI would be beneficial for the interaction of its two DNA-binding domains, positioned at the tips of the V, with the required two of three LacI operators needed for full repression.

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Functional complementation of internal deletion mutants in the lactose permease of Escherichia coli.

Project description:Using the lactose permease of Escherichia coli, a well-characterized membrane protein with 12 transmembrane domains, we demonstrated that certain paired in-frame deletion constructs complement each other functionally. Although cells expressing the deletion mutants individually are unable to catalyze active lactose accumulation, cells simultaneously expressing specific pairs of deletions catalyze transport up to 60% as do cells expressing wild-type permease. Moreover, complementation clearly does not occur at the level of DNA but probably occurs at the protein level. Remarkably, functional complementation is observed only with pairs of permease molecules containing large deletions and is not observed with missense mutations or point deletions. Although the mechanism of functional complementation is obscure, the findings indicate that certain pairs of permease molecules containing specific internal deletions can interact to form a functional complex in the same way phenomenologically as do independently expressed polypeptides corresponding to different N- and C-terminal portions of the permease.

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