Binding of recrystallized and chromatographically purified 8-anilino-1-naphthalenesulfonate to Escherichia coli lac repressor

York, Sheldon S.; Lawson, Robert C.; Worah, Dilip M.

doi:10.1021/bi00614a019

Cited by 49 publications

(30 citation statements)

References 13 publications

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“…Protein concentrations were determined from published molar extinction coefficients. The ammonium salt of ANS was purified by recrystallization and gel filtration on Sephadex LH-20 using water as eluant (York et al, 1978).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease

et al. 1993

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Titration of a salt-free solution of native staphylococcal nuclease by HCl leads to an unfolding transition in the vicinity of pH 4, as determined by near-and far-UV circular dichroism. At pH 2-3, the protein is substantially unfolded. The addition of further HCI results in a second transition, this one to a more structured species (the A state) with the properties of an expanded molten globule, namely substantial secondary structure, little or no tertiary structure, relatively compact size as determined by hydrodynamic radius, and the ability to bind the hydrophobic dye I-anilino-%naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid-unfolded state at pH 2 also causes a transition leading to the A state. Fourier transform infrared analysis of the amide I band was used to compare the amount and type of secondary structure in the native and A states.A significant decrease in a-helix structure, with a corresponding increase in 6 or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured states is proposed.

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Section: Methodsmentioning

confidence: 99%

Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease

et al. 1993

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“…Bis-ANS tends to bind exposed hydrophobic surfaces in partially folded intermediates more tightly than both the native and random coil states of proteins (26). Bis-ANS binding is accompanied by an increase in its fluorescence quantum yield, as well as by a blue shift of the fluorescence emission.…”

Section: Folding Of a Model Three-helix Bundle Proteinmentioning

confidence: 99%

Folding Intermediates of a Model Three-helix Bundle Protein

Chapeaurouge

Johansson

Ferreira

2001

Journal of Biological Chemistry

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The stability and equilibrium unfolding of a model three-helix bundle protein, ␣ 3 -1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated. The combined use of these denaturing agents allowed detection of two partially folded states of ␣ 3 -1, as monitored by circular dichroism, intrinsic fluorescence emission, and fluorescence of the hydrophobic probe bis-ANS (4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid). The overall free-energy change for complete unfolding of ␣ 3 -1, determined from GdnHCl unfolding data, is ؉4.6 kcal/mol. The native state is stabilized by ؊1.4 kcal/mol relative to a partially folded pressure-denatured intermediate (I 1 ). Cold denaturation at high pressure gives rise to a second partially (un)folded conformation (I 2 ), suggesting a significant contribution of hydrophobic interactions to the stability of ␣ 3 -1. The free energy of stabilization of the native-like state relative to I 2 is evaluated to be ؊2.5 kcal/mol. Bis-ANS binding to the pressure-and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partially (un)folded states of ␣ 3 -1. The demonstration of folding intermediates of ␣ 3 -1 lends experimental support to a number of recent protein folding simulation studies of other three-helix bundle proteins that predicted the existence of such intermediates. The results are discussed in terms of the significance of de novo designed proteins for protein folding studies.Understanding the mechanisms by which a polypeptide adopts a stable and functional three-dimensional structure still represents a challenging problem (1). The folding of small proteins usually takes place on timescales close to a millisecond or less, and is believed to occur in a highly cooperative fashion without the presence of populated folding intermediates (2-8). However, recent simulation studies have suggested the existence of intermediate states during the folding of a small model three-helix bundle protein (9 -12). Three-helix bundles represent a simple folding motif found in a variety of soluble and membrane proteins, including spectrin (13) and the extramembranous portion of Staphylococcus aureus protein A (14). Using sequence patterns discovered in coiled coils, the synthesis of amphiphilic ␣-helices that self-assemble into three-or fourhelix bundles stabilized by a hydrophobic core has been successfully achieved (15-17). The de novo design of proteins represents a versatile tool to gain insight into the interplay of forces resulting in conformational stability. Artificial proteins are generally less complex than their native counterparts but at the same time retain the features responsible for the folding process.Recently, Johansson and co-workers (18) reported the synthesis and initial characterization of a native-like three-helix bundle protein, designated ␣ 3 -1. The three different helices of this 65-amino acid polypeptide are joined by (glycine) 4 linkers. NMR solution studies revealed a well structured conf...

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“…Bis-ANS binds to hydrophobic domains of proteins (17). Upon non-covalent binding to such domains, the two naphthyl rings of the bis-ANS molecule become oriented in parallel, which brings about a significant increase in fluorescence quantum yield.…”

Section: Figmentioning

confidence: 99%

Folding of a de Novo Designed Native-like Four-helix Bundle Protein

Chapeaurouge¹,

Johansson²,

Ferreira³

2002

Journal of Biological Chemistry

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The folding of a model native-like dimeric four-helix bundle protein, (␣ 2 ) 2 , was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, ⌬G unf ‫؍‬ 14.3 ؎ 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (␣ 2 ) 2 into folded monomers (␣ 2 ) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (␣ 2 ) 2 of ⌬G diss ‫؍‬ 11.4 ؎ 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (⌬G diss ‫؍‬ 10.5 ؎ 0.1 kcal/ mol). Binding of the hydrophobic fluorescent probe 4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid (bis-ANS) to the pressure-and cold-dissociated states of (␣ 2 ) 2 indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (␣ 2 ) 2 can be described by a three-state transition including a monomeric molten globule-like state.

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Binding of recrystallized and chromatographically purified 8-anilino-1-naphthalenesulfonate to Escherichia coli lac repressor

Cited by 49 publications

References 13 publications

Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease

Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease

Folding Intermediates of a Model Three-helix Bundle Protein

Folding of a de Novo Designed Native-like Four-helix Bundle Protein

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