Electrostatic interactions in the acid denaturation of α‐lactalbumin determined by nmr

Kim, Se‐Ho; Baum, Jean

doi:10.1002/pro.5560070908

Cited by 22 publications

(24 citation statements)

References 62 publications

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“…The NMR spectra for HAMLET are distinctly different from those of native and apo α‐lactalbumin, with fewer resolved peaks, yet contain a larger number of signals than the pH 2 molten globule of human or bovine α‐lactalbumin that yield only a few N‐terminal peaks [25–28]. Instead, the 1 H– 15 N HSQC spectra of HAMLET are similar to the pH 2 spectrum for guinea pig α‐lactalbumin [25] with a somewhat larger number of resonances. Resonances from side chain amides appear at 7.5/113 and 6.8/113 ppm, while most other signals from HAMLET are clustered between 7.5 and 8.7 ppm in the 1 H dimension.…”

Section: Resultsmentioning

confidence: 88%

Compact oleic acid in HAMLET

et al. 2005

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HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex between a-lactalbumin and oleic acid that induces apoptosis in tumor cells, but not in healthy cells. Heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of 13 C-oleic acid in HAM-LET, and to study the 15 N-labeled protein. Nuclear Overhauser enhancement spectroscopy shows that the two ends of the fatty acid are in close proximity and close to the double bond, indicating that the oleic acid is bound to HAMLET in a compact conformation. The data further show that HAMLET is a partly unfolded/molten globule-like complex under physiological conditions.

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Section: Resultsmentioning

confidence: 88%

Compact oleic acid in HAMLET

et al. 2005

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“…For example, a significant loss in dispersion in 1 H chemical shifts upon formation of a molten globule, similar to that seen upon unfolding, has been observed for a-lactalbumin and ribonuclease H1~Baum et al, 1989;Chamberlain & Marqusee, 1998!, and pronounced collapse of the chemical shift dispersion of 1 H, 15 N HSQC spectra has been described upon molten globule formation for apomyo- globin Kim & Baum, 1998!. We have previously demonstrated that the far-UV CD spectra of Sac7d indicate maintenance of the native secondary structure in the acid-and salt-induced folded states~McCrary et al, 1998!. Titration of the protein from N to U and then to acid-folded state with decreasing pH in low salt leads to a well-defined isodichroic point in the far-UV CD, indicating only two states, N and Ũ i.e., the acid folded state is identical to N!.…”

Section: Discussionmentioning

confidence: 99%

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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“…Fewer NMR studies have focused on the native state of the protein (8,13,(28)(29)(30)(31). In particular, the dynamics of the native state have not been studied previously.…”

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confidence: 98%

¹⁵N NMR Relaxation Data Reveal Significant Chemical Exchange Broadening in the α-Domain of Human α-Lactalbumin

Bruylants

Redfield

2009

Biochemistry

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Human alpha-lactalbumin (alpha-LA), a 123-residue calcium-binding protein, has been studied using (15)N NMR relaxation methods in order to characterize backbone dynamics of the native state at the level of individual residues. Relaxation data were collected at three magnetic field strengths and analyzed using the model-free formalism of Lipari and Szabo. The order parameters derived from this analysis are generally high, indicating a rigid backbone. A total of 46 residues required an exchange contribution to T(2); 43 of these residues are located in the alpha-domain of the protein. The largest exchange contributions are observed in the A-, B-, D-, and C-terminal 3(10)-helices of the alpha-domain; these residues have been shown previously to form a highly stable core in the alpha-LA molten globule. The observed exchange broadening, along with previous hydrogen/deuterium amide exchange data, suggests that this part of the alpha-domain may undergo a local structural transition between the well-ordered native structure and a less-ordered molten-globule-like structure.

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Electrostatic interactions in the acid denaturation of α‐lactalbumin determined by nmr

Cited by 22 publications

References 62 publications

Compact oleic acid in HAMLET

Compact oleic acid in HAMLET

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

¹⁵N NMR Relaxation Data Reveal Significant Chemical Exchange Broadening in the α-Domain of Human α-Lactalbumin

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Electrostatic interactions in the acid denaturation of α‐lactalbumin determined by nmr

Cited by 22 publications

References 62 publications

Compact oleic acid in HAMLET

Compact oleic acid in HAMLET

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

15N NMR Relaxation Data Reveal Significant Chemical Exchange Broadening in the α-Domain of Human α-Lactalbumin

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¹⁵N NMR Relaxation Data Reveal Significant Chemical Exchange Broadening in the α-Domain of Human α-Lactalbumin