Structure and Dynamics of the Acid-Denatured Molten Globule State of α-Lactalbumin: A Two-Dimensional NMR Study

Andrei T. Alexandrescu, Philip A. Evans, Maureen Pitkeathly, Jean Baum, Christopher M. Dobson

Research output: Contribution to journalArticle

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Abstract

Two-dimensional 1H-NMR spectroscopy has been used to study the acid-denatured molten globule (A-state) of α-lactalbumin. The NMR spectra show that chemical shift dispersion is limited but significantly greater than that expected for a random coil conformation. The small chemical shift dispersion of side-chain resonances in the A-state together with line broadening associated with conformational averaging indicates that most of the long-range tertiary structure in the A-state is likely to be nonspecific. Side-chain resonances in the A-state are generally shifted somewhat upfield of random coil values; this and the observation of a large number of interresidue NOEs, however, indicate that some side-chain interactions, at least at the level of hydrophobic clustering, exist in the A-state. Analysis of NOESY spectra shows no evidence for an ordered structure for either of the two major clusters of aromatic residues which in the native structure make up part of the hydrophobic core of the helical domain of the native protein. A new aromatic cluster in the A-state which results from rearrangement of the side chains of Tyr103, Trp104, and His107 from their native state positions was, however, detected by a number of well-defined interresidue NOE effects. Similar NOE patterns are observed in a peptide corresponding to residues 101–110 of α-lactalbumin in trifluoroethanol, suggesting that the nonnative structure in the 101–110 region of the A-state is not dependent on specific interactions with the rest of the chain. Trapping experiments indicate that amide protons from regions of the sequence which in the native state are helical are among those strongly protected from solvent exchange in the A-state; those from one of the helices (the C helix) were specifically identified. Taken together, these results reinforce a model of the A-state which has stable regions of localized secondary structure but a largely disordered tertiary structure.

Original languageEnglish (US)
Pages (from-to)1707-1718
Number of pages12
JournalBiochemistry
Volume32
Issue number7
DOIs
StatePublished - Jan 1 1993

Fingerprint

Lactalbumin
Chemical shift
Molten materials
Nuclear magnetic resonance
Trifluoroethanol
Acids
Amides
Nuclear magnetic resonance spectroscopy
Cluster Analysis
Conformations
Protons
Spectrum Analysis
Magnetic Resonance Spectroscopy
Observation
Peptides
Proteins
Experiments
Proton Magnetic Resonance Spectroscopy
Protein Domains

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Alexandrescu, Andrei T. ; Evans, Philip A. ; Pitkeathly, Maureen ; Baum, Jean ; Dobson, Christopher M. / Structure and Dynamics of the Acid-Denatured Molten Globule State of α-Lactalbumin : A Two-Dimensional NMR Study. In: Biochemistry. 1993 ; Vol. 32, No. 7. pp. 1707-1718.
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abstract = "Two-dimensional 1H-NMR spectroscopy has been used to study the acid-denatured molten globule (A-state) of α-lactalbumin. The NMR spectra show that chemical shift dispersion is limited but significantly greater than that expected for a random coil conformation. The small chemical shift dispersion of side-chain resonances in the A-state together with line broadening associated with conformational averaging indicates that most of the long-range tertiary structure in the A-state is likely to be nonspecific. Side-chain resonances in the A-state are generally shifted somewhat upfield of random coil values; this and the observation of a large number of interresidue NOEs, however, indicate that some side-chain interactions, at least at the level of hydrophobic clustering, exist in the A-state. Analysis of NOESY spectra shows no evidence for an ordered structure for either of the two major clusters of aromatic residues which in the native structure make up part of the hydrophobic core of the helical domain of the native protein. A new aromatic cluster in the A-state which results from rearrangement of the side chains of Tyr103, Trp104, and His107 from their native state positions was, however, detected by a number of well-defined interresidue NOE effects. Similar NOE patterns are observed in a peptide corresponding to residues 101–110 of α-lactalbumin in trifluoroethanol, suggesting that the nonnative structure in the 101–110 region of the A-state is not dependent on specific interactions with the rest of the chain. Trapping experiments indicate that amide protons from regions of the sequence which in the native state are helical are among those strongly protected from solvent exchange in the A-state; those from one of the helices (the C helix) were specifically identified. Taken together, these results reinforce a model of the A-state which has stable regions of localized secondary structure but a largely disordered tertiary structure.",
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Structure and Dynamics of the Acid-Denatured Molten Globule State of α-Lactalbumin : A Two-Dimensional NMR Study. / Alexandrescu, Andrei T.; Evans, Philip A.; Pitkeathly, Maureen; Baum, Jean; Dobson, Christopher M.

In: Biochemistry, Vol. 32, No. 7, 01.01.1993, p. 1707-1718.

Research output: Contribution to journalArticle

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T1 - Structure and Dynamics of the Acid-Denatured Molten Globule State of α-Lactalbumin

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AB - Two-dimensional 1H-NMR spectroscopy has been used to study the acid-denatured molten globule (A-state) of α-lactalbumin. The NMR spectra show that chemical shift dispersion is limited but significantly greater than that expected for a random coil conformation. The small chemical shift dispersion of side-chain resonances in the A-state together with line broadening associated with conformational averaging indicates that most of the long-range tertiary structure in the A-state is likely to be nonspecific. Side-chain resonances in the A-state are generally shifted somewhat upfield of random coil values; this and the observation of a large number of interresidue NOEs, however, indicate that some side-chain interactions, at least at the level of hydrophobic clustering, exist in the A-state. Analysis of NOESY spectra shows no evidence for an ordered structure for either of the two major clusters of aromatic residues which in the native structure make up part of the hydrophobic core of the helical domain of the native protein. A new aromatic cluster in the A-state which results from rearrangement of the side chains of Tyr103, Trp104, and His107 from their native state positions was, however, detected by a number of well-defined interresidue NOE effects. Similar NOE patterns are observed in a peptide corresponding to residues 101–110 of α-lactalbumin in trifluoroethanol, suggesting that the nonnative structure in the 101–110 region of the A-state is not dependent on specific interactions with the rest of the chain. Trapping experiments indicate that amide protons from regions of the sequence which in the native state are helical are among those strongly protected from solvent exchange in the A-state; those from one of the helices (the C helix) were specifically identified. Taken together, these results reinforce a model of the A-state which has stable regions of localized secondary structure but a largely disordered tertiary structure.

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