Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins.

Spotts, Ronit; Chakerian, Artemis E.; Matthews, Kathleen S.

doi:10.1016/s0021-9258(18)54453-7

Cited by 27 publications

(13 citation statements)

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“…Note that V52A, V52H, V52S, and V52W retain significant affinity in the presence of inducer. filter binding was used to monitor decreased levels of bound DNA as a function of inducer concentration (30,31,38,39).…”

Section: Resultsmentioning

confidence: 99%

“…In operator release experiments, constant concentrations of protein (at ∼80% saturation for operator binding in the absence of inducer; see figure legends) were incubated with ∼2 × 10 -12 M operator to form the LacI-DNA complex; then IPTG (varied from ∼10 -8 to 10 -4 M) was added to the reaction to release operator (30,31,38,39). Again, the retained operator was quantified using a Fuji phosphorimager.…”

Section: T H Imentioning

confidence: 99%

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Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

2006

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A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short α-helices when bound to DNA, but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge helix folding and/or hinge•hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality. KeywordsLacI; helical propensity; allostery; DNA binding Protein folding has long been a fundamental issue in biological sciences. Beyond the general question of how a polypeptide sequence adopts a three-dimensional structure, coupled folding and binding have been identified as a key feature of partially unstructured proteins in multiple regulatory processes (e.g., transcription regulation, signal transduction, membrane transport and signaling (6-9)). Indeed, more and more intrinsically disordered proteins are found to fold upon binding to their target partners, ligands, or even small ions (10-13). In the case of transcription regulation, mutual cooperative folding of both protein and DNA has been † This work was supported by NIH Grant GM22441 and Robert A. Welch Grant C-576 to Kathleen Shive Matthews. Liskin Swint-Kruse is supported by NIH Grant P20 RR17708 from the Institutional Development Award program of the National Center for Research Resources.*To whom correspondence should be addressed. Telephone: 713−348−4871; Fax: 713−348−6149; Email: E-mail: ksm@bioc.rice.edu.. observed (6,11,14,15). In this paper, coupled folding is explored in greater detail for the hinge helix of the lactose repressor protein (LacI) 1 . NIH Public AccessLacI is a premier model for the regulation of gene expression and allosteric transition in biological systems (2) 2 . This 150-kDa homotetramer is a dimer of functionally independent dimers, with ...

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Section: Resultsmentioning

confidence: 99%

Section: T H Imentioning

confidence: 99%

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

2006

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“…In the purine repressor structure, Asp275 participates in hydrogen bond and charge interactions with Arg196, which anchors Asp275 interaction with ligand (Schumacher et al, 1994). Asp274 in lac repressor might function similarly, with its side chain interacting with that of Arg197, a residue at which substitution significantly alters inducer affinity (Spotts et al, 1991). However, it should be underscored that differences in the arrangement of side chains in these homologous proteins are required to provide the basis for distinctions in their binding and functional properties, and the presence of similar side chains does not ensure similar arrangement in binding sites specific for different ligands (i.e., purine vs sugar).…”

Section: Discussionmentioning

confidence: 99%

Role of Asp274 in lac Repressor: Diminished Sugar Binding and Altered Conformational Effects in Mutants

Chang

Matthews²

1995

Biochemistry

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The role of Asp274 in inducer binding of lac repressor has been explored by spectroscopic measurements, fluorescence quenching, in vitro induction assays, and chemical modification of mutants with conservative substitutions at this site. Although no fluorescence emission shift or characteristic UV difference spectrum was observed at high inducer concentration, fluorescence quenching, effects on operator binding, and chemical modification results indicate indirectly that the mutants Asp274-->Asn and Asp274-->Glu bind sugar, albeit with very low affinity (> 0.1 M). Consistent with very weak inducer binding indicated by protection from fluorescence quenching by iodide, operator binding activity of these two mutant proteins is altered at very high IPTG concentration, although in opposite directions. The distinct effects of inducer on operator binding in these two mutant proteins as well as substantial differences in the effect of sugar ligand on chemical modification of Cys107 and Cys140 by 2-(bromoacetamido)-4-nitrophenol suggest that the conformation of the protein before and after association with sugar may differ in these mutant proteins. Fluorescence quenching assays of lac mutant proteins at Asp274 indicate the proximity of Trp220 to the side chain at position 274, consistent with the location of this residue in the structural model of lac repressor and in the crystallographic structure of the homologous purine repressor. From these results, we conclude that Asp274 is in the inducer binding site, that the character of this residue is crucial to inducer binding, and that interaction of sugar with the side chain at this position may be associated with the conformational change necessary for generating high affinity ligand binding.

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“…Curious about the thermodynamic implications of inducer binding on LacI function, the laboratory of Mitchell Lewis next sought to reengineer their pair of heterodimeric LacI repressor proteins to incorporate combinations of functionally active and inactive inducer binding sites (i.e., 0, 1, and 2 functionally active binding sites, Figure ) . Again, referencing mutational data made available from the early structure–function studies of the LacI allowed Lewis and colleagues to produce inducer insensitive (I S ) LacI incapable of binding inducer molecules using the single point mutations R197A or R197G . Using their GFP reporter system, Daber et al observed that their heterodimer (LacI Y282S + Het2S) containing two functional IPTG binding sites displayed a similar repression‐induction profile to that of WT LacI, with expression measured at 76% under induction relative to reporter gene expression in the absence of repressor.…”

Section: Engineering Alternate Functions and Logical Gatingmentioning

confidence: 99%

Deconstruction of complex protein signaling switches: a roadmap toward engineering higher‐order gene regulators

Davey

Wilson

2017

WIREs Nanomed Nanobiotechnol

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The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene-function analysis, and protein manufacturing. The most successful approaches to date are based on modulating messenger RNA (mRNA) synthesis via their inducible coupling to transcriptional effectors, which requires biosensing functionality. A hallmark of biological sensing is the conversion of an exogenous signal, usually a small molecule or environmental cue such as a protein-ligand interaction, into a useful output or response. One of the most utilized regulatory proteins is the lactose repressor (LacI). In this review we will (1) explore the mechanochemical structure-function relationship of LacI; (2) discuss how the physical attributes of LacI can be leveraged to identify and understand other regulatory proteins; (3) investigate the designability (tunability) of LacI; (4) discuss the potential of the modular design of novel regulatory proteins, fashioned after the topology and mechanochemical properties of LacI. WIREs Nanomed Nanobiotechnol 2017, 9:e1461. doi: 10.1002/wnan.1461 For further resources related to this article, please visit the WIREs website.

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Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins.

Cited by 27 publications

References 49 publications

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

Role of Asp274 in lac Repressor: Diminished Sugar Binding and Altered Conformational Effects in Mutants

Deconstruction of complex protein signaling switches: a roadmap toward engineering higher‐order gene regulators

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