An operator-induced conformational change in the C-terminal domain of the lambda repressor.

Saha, Rina; Banik, Utpal; Bandopadhyay, Sandip; Mandal, Nripendranath; Bhattacharyya, Bhabatarak; Roy, Siddhartha

doi:10.1016/s0021-9258(18)42633-6

Cited by 31 publications

(16 citation statements)

References 31 publications

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“…Experiments with ANS provide a third line of evidence for a pH-regulated structural change in class II molecules. ANS is an environment-sensitive fluorescent probe that has been extensively used to monitor conformation changes in proteins (40)(41)(42)(43)(44). The ANS fluorescence intensity was markedly increased with an associated blue shift in ~ ..... in the presence of detergent-solubilized IA d, IA k, IE d, and IE k molecules at pH 5 compared with 7 (Figs.…”

Section: Discussionmentioning

confidence: 99%

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A structural transition in class II major histocompatibility complex proteins at mildly acidic pH.

Runnels

Moore

Jensen

1996

The Journal of Experimental Medicine

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SummaryPeptide binding by class II major histocompatibility complex proteins is generally enhanced at low pH in the range of hydrogen ion concentrations found in the endosomal compartments of antigen-presenting cells. We and others have proposed that class II molecules undergo a reversible conformational change at low pH that is associated with enhanced peptide loading. However, no one has previously provided direct evidence for a structural change in class II proteins in the mildly acidic pH conditions in which enhanced peptide binding is observed. In this study, susceptibility to denaturation induced by sodium dodecyl sulfate (SDS) detergent or heat was used to probe the conformation of class II at different hydrogen ion concentrations. Class II molecules became sensitive to denaturation at pH 5.5-6.5 depending on the allele and experimental conditions. The observed structural transition was fully reversible if acidic pH was neutralized before exposure to SDS or heat. Experiments with the environment-sensitive fluorescent probe ANS (8-anilino-l-naphthalene-sulfonic acid) provided further evidence for a reversible structural transition at mildly acidic pH associated with an increase in exposed hydrophobicity in class II molecules. IA d conformation was found to change at a higher pH than IE d, IE k, or IA k, which correlates with the different pH optimal for peptide binding by these molecules. We conclude that pH regulates peptide binding by influencing the structure of class II molecules.

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Section: Discussionmentioning

confidence: 99%

“…ANS is an environment-sensitive fluorescent probe that is used to monitor structural changes in proteins (40)(41)(42)(43)(44). The emission characteristics of ANS vary with the polarity of its environment.…”

Section: N S Fluorescence Studies Demonstrate a Reversible Structural Transition In Class II Molecules At Mildly Acidic Phmentioning

confidence: 99%

A structural transition in class II major histocompatibility complex proteins at mildly acidic pH.

Runnels

Moore

Jensen

1996

The Journal of Experimental Medicine

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“…Fluorescence emission intensities (at 485 nm) obtained from titrations with bis-ANS and DnaK were used for Scatchard and Hill plot fitting (Cantor & Schimmel, 1980). Based on the plot fitting, the number of molecules (n) of bis-ANS bound to DnaK and the dissociation constants (K<1) for both DnaK-(N)-bis-ANS and DnaK(I)-bis-ANS complexes were estimated as described below (Semisotnov et al, 1991;Bhattacharyya et al, 1991;Lee et al, 1991;Saha et al, 1992). For solutions of bis-ANS with concentration higher than 60 µ , the fluorescence intensities were corrected for the inner filter effect (Horowitz & Criscimagna, 1985;Calhun et al, 1983;Lakowicz, 1983).…”

Section: Methodsmentioning

confidence: 99%

Protein Conformational Changes Induced by 1,1'-Bis(4-anilino-5-naphthalenesulfonic acid): Preferential Binding to the Molten Globule of DnaK

Shi¹,

Palleros²,

Fink³

1994

Biochemistry

131

102

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1,1'-Bis(4-anilino-5-naphthalenesulfonic acid) (bis-ANS), a hydrophobic fluorescent molecular probe which has been shown to bind to compact intermediate states of proteins (molten globules) and also to many nucleotide binding sites, induces a conformational change in DnaK by preferentially binding to its partially folded intermediate state (I) and thus shifting the equilibrium from favoring the native state (N) to favoring the I state. The conformational change was detected by CD, fluorescence emission, size exclusion chromatography, and small-angle X-ray scattering. The presence of bis-ANS significantly decreases the midpoint, Tm, of the initial transition (N-->I) in the thermal unfolding of DnaK, resulting in the apparent destabilization of the native state of DnaK. There is a linear correlation between the apparent free energy (reflected by Tm) of this transition and the concentration of bis-ANS. Bis-ANS does not affect the midpoint of the transition for DnaK from the intermediate to the unfolded state (U). An additional small transition from I to I*, a more expanded intermediate state, was observed, suggesting that the thermal denaturation of DnaK proceeds via a four-state (N-->I-->I*-->U) unfolding process. The addition of nucleotides, ADP or ATP, to the DnaK-bis-ANS complex causes a decrease in bis-ANS fluorescence emission due to the release of bound bis-ANS from the intermediate state of DnaK. This is due to preferential binding of the nucleotide to the native state of DnaK, resulting in a shift in the equilibrium from the intermediate toward the native state rather than the direct displacement of bis-ANS bound in the nucleotide binding site. Denaturation of DnaK induced by bis-ANS can be minimized by working at a temperature much lower than the Tm of the protein, at low dye concentration, and in the presence of nucleotide. Under these conditions, bis-ANS binds to the native state of DnaK.

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“…A-Repressor was isolated from a strain of Escherichia coli RR1 15 lac Z carrying the plasmid pEA 305 which contains the wild-type cl gene under the control of the tac promoter. The details of the purification procedure were given in Saha et al (1992). The native repressor concentration was determined using Ex%28o = 11.3.…”

Section: Methodsmentioning

confidence: 99%

Role of the C-Terminal Tail Region in the Self-Assembly of .lambda.-Repressor

Bandyopadhyay¹,

Banik²,

Bhattacharyya³

et al. 1995

Biochemistry

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Acrylamide quenching of the tryptophan fluorescence of the lambda-repressor at different protein concentrations indicates that one of the three tryptophan residues, W129, W142, and W230, undergoes a change in environment upon self-assembly, from dimer to associated species. Quenching data suggest that this tryptophan residue is inaccessible to low concentrations of acrylamide and is blue-shifted in the associated form. In the dimer, this tryptophan residue is highly accessible to acrylamide and is red-shifted. NBS oxidation, at protein concentrations which favor the associated form, showed that this tryptophan is also significantly protected from NBS oxidation. HPLC peptide mapping of NBS-oxidized lambda-repressor, amino acid analysis, and sequencing indicate that the protected, blue-shifted tryptophan is tryptophan 230. A mutant repressor (F235C) was specifically labeled at Cys 235 with an environment-sensitive probe, acrylodan. The acrylodan fluorescence of the labeled F235C lambda-repressor undergoes a significant blue-shift, accompanied by fluorescence enhancement, upon protein association. Along with other genetic evidence, these results suggest involvement of the C-terminal tail region in the self-assembly of the lambda-repressor.

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An operator-induced conformational change in the C-terminal domain of the lambda repressor.

Cited by 31 publications

References 31 publications

A structural transition in class II major histocompatibility complex proteins at mildly acidic pH.

A structural transition in class II major histocompatibility complex proteins at mildly acidic pH.

Protein Conformational Changes Induced by 1,1'-Bis(4-anilino-5-naphthalenesulfonic acid): Preferential Binding to the Molten Globule of DnaK

Role of the C-Terminal Tail Region in the Self-Assembly of .lambda.-Repressor

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