Binding of naphthalene dyes to the N and A conformers of bovine α-lactalbumin

Mulqueen, Paul; Kronman, Martin J.

doi:10.1016/0003-9861(82)90275-2

Cited by 87 publications

(46 citation statements)

References 17 publications

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“…The fluorescence enhancement of the dye ANS is also typically used as an indicator of a molten globule state~Mulqeen & Kronman, 1982;Goto & Fink, 1989;Semisotnov et al, 1991;Fink et al, 1994!, presumably due to interactions with hydrophobic patches~Stryer, 1965!. Typically, the fluorescence is unchanged in the presence of the native state, while the unfolded state leads to some enhancement, e.g., sixfold in parvalbumiñ Fink et al, 1994!…”

Section: Discussionmentioning

confidence: 99%

“…and as a diagnostic indicator of the collapsed, molten globule state~Mulqeen & Kronman, 1982;Semisotnov et al, 1991!. Although ANS binds to proteins through electrostatic interactions~Matulis & Lovrien, 1998!, the fluorescence of ANS is enhanced by the presence of exposed hydrophobic regions. The fluorescence of ANS was unaffected by native Sac7d at pH 7~Fig.…”

Section: Ans Fluorescencementioning

confidence: 99%

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The acid‐induced folded state of Sac7d is the native state

et al. 2000

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Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt McCrary BS, Bedell J, Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224!. Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid-and salt-induced folded states have a native fold and are not molten globular.1 H, 15 N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1 H and 15 N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1 H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid-and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.Keywords: anion binding; ANS binding; circular dichroism; hyperthermophile; molten globule; NMR; small-angle X-ray scattering; Sulfolobus Proteins are commonly destabilized by acid with maximal unfolding typically occurring near pH 2~Linderström- Lang, 1924;Tanford, 1970;Matthew et al., 1985;Yang & Honig, 1993;Oliveberg et al., 1994;Garcia-Moreno, 1995!. In many cases, a further decrease in pH, or the addition of salt, leads to a collapse of the unfolded chain into a compact "denatured" state referred to as the acid or A-state~Goto et al., 1990;Fink et al., 1994!. The A-state is stabilized by anions contributed from either acid or salt. It has been shown to exist for a diverse array of proteins ranging from nearly all a-helical to largely all b-sheet~Fink et al., 1994!. In every instance described to date, the properties of the A-state have been those of the so-called molten globule~Ptitsyn, 1987globule~Ptitsyn, , 1995Kuwajima, 1989;Fink et al., 1994; Fink, 1995!, i.e., a folded intermediate with native-like secondary structure as indicated by the far-UV CD spectrum, but an expanded structure with a radius of gyration approximately 10-20% greater than that of the native state, loss of close packing tertiary contacts as indicated by the absence of near-UV CD and NMR chemical shift dispersion, and exposure of hydrophobic patches as indicated by enhanced fluorescence of the dye ANS. T...

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Section: Discussionmentioning

confidence: 99%

Section: Ans Fluorescencementioning

confidence: 99%

The acid‐induced folded state of Sac7d is the native state

et al. 2000

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“…ANS binds to the solvent-exposed hydrophobic surfaces that are found in partially unfolded proteins (24). CE can readily be used to screen for ANS binding to the ␤ 2 m f and ␤ 2 m s species because ANS is a sulfonated molecule, and separations by CE are very sensitive to changes in analyte charge/mass ratios (25).…”

Section: Conformational Variant Of ␤ 2 M Has An Increased Binding Affmentioning

confidence: 99%

Conformational Intermediate of the Amyloidogenic Protein β2-Microglobulin at Neutral pH

Heegaard¹,

Sen²,

Kaarsholm³

et al. 2001

Journal of Biological Chemistry

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Aggregation and fibrillation of ␤ 2 -microglobulin are hallmarks of dialysis-related amyloidosis. We characterize perturbations of the native conformation of ␤ 2 -microglobulin that may precede fibril formation. For a ␤ 2 -microglobulin variant cleaved at lysine 58, we show using capillary electrophoresis that two conformers spontaneously exist in aqueous buffers at neutral pH. Upon treatment of wild-type ␤ 2 -microglobulin with acetonitrile or trifluoroethanol, two conformations were also observed. These conformations were in equilibrium dependent on the sample temperature and the percentage of organic solvent present. Circular dichroism showed a loss of ␤-structures and gain of ␣-helices. Reversal to the native conformation occurred when removing the organics. Affinity capillary electrophoresis experiments showed increased specific interactions of the nonnative ␤ 2 -microglobulin conformation with the dyes 8-anilino-1-naphthalene sulfonic acid and Congo red. The observations may relate to early folding events prior to amyloid fibrillation and facilitate the development of methods to detect and inhibit pro-amyloid protein and peptide conformations.Abnormal protein folding leads to amyloid deposition in a number of different chronic disorders including Alzheimer's disease, senile systemic amyloidosis, transmissible spongiform encephalopathies, and dialysis-related amyloidosis (1). Different proteins and polypeptides can acquire the abnormal conformational changes that lead to amyloid formation, and at least 19 different proteins are now known to be involved in the generation of amyloid in vivo (1). In the case of dialysis-related amyloidosis the protein involved is ␤ 2 -microglobulin (␤ 2 m), 1 which aggregates as amyloid fibrils in joints and tissues in ϳ20% of patients with kidney failure within 2 years after the onset of hemodialysis treatment (2). This type of amyloidosis does not seem to require a mutated, processed, or chemically modified precursor protein. Although the cause of the abnormal folding in vivo is unknown, it has been reported recently that ␤ 2 m can be induced to generate amyloid fibrils in vitro at low pH and high salt conditions (3) and that fibrillar ␤ 2 m can be refolded back in vitro (4).Similar pre-amyloid folding states have now been demonstrated for a number of amyloidosis-related proteins (5-9) and also in other proteins and peptides without known amyloid disease associations (10 -13). ␤ 2 m is an attractive protein for studying amyloid-promoting folding events because it is small (M r ϭ 11,729), nonglycosylated, nonpolymorphic, and has a simple native structure (14), i.e. it is a one-chain all ␤-protein with two antiparallel pleated sheets of ␤-strands linked together by a disulfide bridge (15). It is structurally homologous to constant domains of immunoglobulins (14). In chronic renal failure ␤ 2 m serum concentrations may increase 10 -70 times despite dialysis, but there is no simple relationship between ␤ 2 m serum concentrations and the extent of amyloidosis (16 The Lys 58 -␤ 2 ...

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“…For example, apo-␣-LA binds more effectively to model membranes (9) and possesses higher affinity for hydrophobic probes than does Ca 2ϩ -␣-LA (7,13,22). Furthermore, under acidic conditions (molten globule), ␣-LA associates readily with these lipophiles (23,24).…”

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confidence: 99%