Browsing by Author "Zhan, Hongli"

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    Biophysical characterization of the allosteric transition in lactose repressor protein (LacI)
    (2005) Zhan, Hongli; Matthews, Kathleen S.
    Allosteric transition, the basis of signal transduction and central to the function of regulatory proteins (e.g., transcriptional factors), is widely involved in biological systems with conformational change as a key characteristic. Although end-state structures are known for many proteins, less is known about the underlying detailed mechanism of the allosteric transition. We have used LacI as a model system to investigate this process at the atomic level. The work in this thesis focuses on three regions of LacI: the core pivot region, the N-subdomain monomer-monomer interface, and the hinge region. Characterization of representative mutants (L148F, S151P, P320A, and Q60G/L148F) demonstrated that the core pivot region exerts long-range effects on LacI function. For L148F and S151P, operator and inducer binding are altered in an inverse fashion with binding for one ligand strengthened, and binding for the other ligand weakened. Further characterization of L148F and S 151P has indicated that the conformational equilibrium is shifted towards the induced state in L148F and towards the repressed end in S151P. This conclusion is supported by detailed thermodynamic ligand binding assays and UV difference spectra. Detailed unfolding/refolding studies further suggest that the intrinsic ligand-binding properties of L148F and S151P are altered. Global fitting of all ligand-binding data is underway to further characterize these shifts. Our data for K84 hydrophobic variants (K84A/L) disclose impeded allosteric response to inducer, a state that is supported by a unique pattern in UV difference spectra. Operator release kinetics for K84A/L in response to IPTG suggest that two inducer molecules are required to release operator DNA. Characterization of 13 substitutions at V52, including binding to operator sequence variants, indicates a dominant role of the protein-operator interaction in LacI allostery and high affinity operator binding. Moreover, subsets of mutants that decouple inducer binding and conformational change were identified. In summary, this thesis work emphasizes the key role of several regions in LacI allostery, identifies several LacI allosteric intermediates, and discloses intermediates trapped along the allosteric pathway by mutation that correlate with points along the TMD simulation.
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    Lactose repressor hinge domain independently binds DNA
    (Wiley, 2018) Xu, Joseph S.; Hewitt, Madeleine N.; Gulati, Jaskeerat S.; Cruz, Matthew A.; Zhan, Hongli; Liu, Shirley; Matthews, Kathleen S.
    The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ∼40° and aligns flanking semi-symmetric DNA sites for optimal contact by the N-terminal helix-turn-helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ∼4-fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.