<i>lac</i>‐Repressor headpiece constitutes a reversibly unfolding domain

Hinz, Hans-Ju ̈rgen; Cossmann, Matthias; Beyreuther, Konrad

doi:10.1016/0014-5793(81)80175-5

Cited by 9 publications

(4 citation statements)

References 15 publications

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“…The decrease in Kq at higher temperature could also be due to thermal denaturation of the headpiece. Thus, the Tm of 2: 40 °C (Hinz et al, 1981; Schnarr & Maurizot, 1982) for the headpiece is close enough to the temperature ( > 25 °C) at which Kd increases with temperature. That both native repressor and headpiece both show a similar nonlinear dependence of the dissociation constant with temperature rules out the trivial explanation that the effect for the whole repressor is possibly attributable to the dissociation of individual subunits.…”

Section: Resultsmentioning

confidence: 66%

Phosphorus-31 nuclear magnetic resonance spectra and dissociation constants of lac repressor headpiece.cntdot.duplex operator complexes: The importance of phosphate ester backbone flexibility in protein-DNA recognition

Botuyan¹,

Keire²,

Kroen³

et al. 1993

Biochemistry

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An alkaline phosphatase assay was used to determine the dissociation constants (KD) of the lac repressor N-terminal 56 amino acid fragment of the wild type and of a Y7I mutant complexed to 22 base pair (bp) wild-type and mutant symmetrical operator sequences. KD's in 0.35 M monovalent salt ranged from 5.4 X 10(-8) M for the wild-type repressor.wild-type operator complex to approximately > 1 X 10(-6) M for the wild-type repressor.nonspecific DNA complex. Mutant operators O2 (G5 --> A5 and G16 --> T16) and O4 (G5 --> C5 and C16 --> G16) bind nearly as tightly as the wild-type headpiece, while mutant O3 (A8 --> T8 and T13 --> A13) binds over 5-fold poorer. Operators O1, O2, and O4 bind ca. 10-fold poorer to the Y7I mutant headpiece. Operator O3 binds 2-fold poorer to the mutant headpiece. The temperature and salt dependence on the dissociation constants of wild-type headpiece binding to 22-bp operator support the conclusion that the headpiece contains the major DNA recognition portion of the protein and that electrostatics plays as important a role in the binding of operator to headpiece as it does in the whole repressor. The 31P NMR spectra of shortened 14-bp wild-type and mutant symmetrical operators bound to the N-terminal 56-residue headpiece of the Y7I mutant repressor were compared to the spectra of the same operator bound to the wild-type repressor headpiece. These results are consistent with a recent proposal [Karslake, C., Botuyan, M. V., & Gorenstein, D. G. (1992) Biochemistry 31, 1849-1858] that specific, tight-binding operator.protein complexes retain the inherent phosphate ester conformational flexibility of the operator itself, whereas the phosphate esters are conformationally restricted in the weak-binding operator-protein complexes. This retention of backbone torsional freedom in tight complexes is entropically favorable and provides a mechanism for protein discrimination of different operator binding sites.

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Section: Resultsmentioning

confidence: 66%

Phosphorus-31 nuclear magnetic resonance spectra and dissociation constants of lac repressor headpiece.cntdot.duplex operator complexes: The importance of phosphate ester backbone flexibility in protein-DNA recognition

Botuyan¹,

Keire²,

Kroen³

et al. 1993

Biochemistry

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“…The large influence of the coenzyme PLP on the thermodynamic parameters of subunit association has been demonstrated in a previous study (Wiesinger et al, 1979). It has been found that proton flux, reaction enthalpies, and heat capacity changes, which are indicative of structural changes of the macromolecule (Sturtevant, 1977;Hinz et al, 1981;Hinz, 1983), are very different for apoenzyme and holoenzyme formation from the isolated a and ß2 and a and (jd-PLP)2 subunits, respectively.…”

mentioning

confidence: 72%

Energetic adaptation of ligand binding to subunit structure of tryptophan synthase from Escherichia coli

Wiesinger¹,

Hinz²

1984

Biochemistry

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The binding of indole and L-serine to the isolated alpha and beta 2 subunits and the native alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli was investigated by direct microcalorimetry to reveal the energetic adaptation of ligand binding to the subunit structure of a multienzyme complex. In contrast to the general finding that negative heat capacity changes are associated with ligand binding to proteins, complex formation of indole and the alpha subunit involves a small positive change in heat capacity. This unusual result was considered as being indicative of a loosening of the protein structure. Such an interpretation is in good agreement with results of chemical accessibility studies (Freedberg & Hardman, 1971). Whereas the thermodynamic parameters of indole binding are not influenced by the subunit interaction, the large negative change in heat capacity of -6.5 kJ/(K X mol of beta 2) measured for the binding of L-serine to the isolated beta 2 subunit disappears completely when serine interacts with the tetrameric complex. These data demonstrate that the energy transduction pattern and therefore the functional roles of the substrates indole and L-serine vary strongly with the subunit structure of tryptophan synthase.

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“…Our analysis of the purine repressor structure indicates that formation of the antiparallel hinge helices buries $2000 A Ê 2 of non-polar surface and $1600 A Ê 2 polar surface, comparable to that removed in the interface, contributing $ À 500 cal mol À1 K À1 to the overall ÁC of HP folding is À300 cal mol À1 K À1 (Hinz et al, 1981), and the ellipticity of the unbound HP corresponds to $30% alpha-helix, half of that observed in the half-site complex (Chuprina et al, 1993 , and that a total of $70 residues fold. From the consistency between this calculation and the experimentally determined value of ÁC o obs and the predicted number of residues which fold (90 AE 10, see Table 4), we propose that the surface area buried in folding transitions in the hinge helices and the HP dominates the ÁC (Takeda et al, 1992).…”

Section: Interpretation Of Heat Capacity Changes Of Protein-nucleic Amentioning

confidence: 95%

“…However, [salt] is known to have profound effects on the stability of the HP (Wemmer et al, 1981;Schnarr & Maurizot, 1982); at neutral pH the denaturation temperature increases from 310 K at low [salt] ($10 mM) to 340 K at high [salt] (1 M), and isolation of intact HP from the core domain by limited proteolytic digestion is only possible at high [salt] (1 M Tris-HCl). Even at relatively high [salt] (50.4 M) and neutral pH, the HP structure appears to be``loose'' or``mobile'' when compared to other highly ordered proteins: its amide protons exchange rapidly (Wemmer et al, 1981), NMR resonance lines are sharp (WadeJardetsky et al, 1979), and its thermally induced unfolding transition is broad (Hinz et al, 1981;Schnarr & Maurizot, 1981, 1982Wemmer et al, 1981). Under these conditions, interactions which stabilize the HP appear only weakly cooperative, in contrast to the highly cooperative folding units of other proteins of comparable size (see for example Alexander et al, 1992).…”

Section: Interpretation Of Heat Capacity Changes Of Protein-nucleic Amentioning

confidence: 97%

Thermodynamics of the interactions of Lac repressor with variants of the symmetric Lac operator: effects of converting a consensus site to a non-specific site

et al. 1997

Journal of Molecular Biology

112

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lac‐Repressor headpiece constitutes a reversibly unfolding domain

Cited by 9 publications

References 15 publications

Phosphorus-31 nuclear magnetic resonance spectra and dissociation constants of lac repressor headpiece.cntdot.duplex operator complexes: The importance of phosphate ester backbone flexibility in protein-DNA recognition

Phosphorus-31 nuclear magnetic resonance spectra and dissociation constants of lac repressor headpiece.cntdot.duplex operator complexes: The importance of phosphate ester backbone flexibility in protein-DNA recognition

Energetic adaptation of ligand binding to subunit structure of tryptophan synthase from Escherichia coli

Thermodynamics of the interactions of Lac repressor with variants of the symmetric Lac operator: effects of converting a consensus site to a non-specific site

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