title: Functional impact of polar and acidic substitutions in the lactose repressor hydrophobic monomer.monomer interface with a buried lysine.
abstract: Despite predicted energetic penalties, the charged K84 side chains of tetrameric lactose repressor protein (LacI) are found buried within the highly hydrophobic monomer.monomer interface that includes side chains of V94 and V96. Once inducer binding has occurred, these K84 side chains move to interact with the more solvent-exposed side chains of D88 and E100'. Previous studies demonstrated that hydrophobic substitutions for K84 increased protein stability and significantly impaired the allosteric response. These results indicated that enhanced hydrophobic interactions at the monomer.monomer interface remove the energetic driving force of the buried charges, decreasing the likelihood of a robust conformational change and stabilizing the structure. We hypothesized that creating a salt bridge network with the lysine side chains by including nearby negatively charged residues might result in a similar outcome. To that end, acidic residues, D and E, and their neutral amides, N and Q, were substituted for the valines at positions 94 and 96. These variants exhibited one or more of the following functional changes: weakened inducer binding, impaired allosteric response, and diminished protein stability. For V96D and V96E, ion pair formation with K84 appears optimal, and the loss of inducer response exceeds that of the hydrophobic K84A and -L variants. However, impacts on functional properties indicate that stabilizing the buried positive charge with polar or ion pair interactions is not functionally equivalent to structural stabilization via hydrophobic enhancement.
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA. zhanhl@rice.edu
authors.forename: Hongli
authors.initials: H
authors.lastname: Zhan
authors.forename: Zhifei
authors.initials: Z
authors.lastname: Sun
authors.forename: Kathleen Shive
authors.initials: KS
authors.lastname: Matthews
mesh_terms: D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics*; D024342:Amino Acids, Acidic / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D005453:Fluorescence; D057927:Hydrophobic and Hydrophilic Interactions; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D056968:Lac Repressors; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009875:Operator Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
chemical_list: D024342:Amino Acids, Acidic; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D056968:Lac Repressors; D050505:Mutant Proteins; D021122:Protein Subunits; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D014508:Urea; D008239:Lysine