Mutations Can Cause Large Changes in the Conformation of a Denatured Protein

John Flanagan, Mikio Kataoka, Tetsuro Fujisawa, Donald M. Engelman

Research output: Contribution to journalArticle

108 Citations (Scopus)

Abstract

Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WT SNaseA) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936–944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748–752]. Because of these and other properties of the SNaseΔ polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNaseΔ may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNaseΔ, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNaseΔ), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNaseΔ exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNaseΔ were all of the m− type (m− substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNaseΔ that were more extended and coil-like than WT SNaseΔ were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNaseΔ, as well as both the m+ and m− substituted versions of SNaseΔ, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain. The correspondence between changes in Rg produced by amino acid substitutions in SNaseΔ and the size of the denatured state predicted on the basis of solvent denaturation studies for these mutated forms supports the view that some amino acid substitutions can affect stability by changing the average conformation of the denatured state. In addition, our results are consistent with the idea that secondary structure formation and chain condensation occur simultaneously in this system.

Original languageEnglish (US)
Pages (from-to)10359-10370
Number of pages12
JournalBiochemistry
Volume32
Issue number39
DOIs
StatePublished - Jan 1 1993

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Amino Acid Substitution
Conformations
Substitution reactions
Peptides
Mutation
Polymers
Proteins
Amino Acids
Small Angle Scattering
Micrococcal Nuclease
Denaturation
Circular Dichroism
Ribosomes
Biochemistry
Scattering
Spectrum Analysis
Hot Temperature
X-Rays
Circular dichroism spectroscopy
Temperature

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Flanagan, John ; Kataoka, Mikio ; Fujisawa, Tetsuro ; Engelman, Donald M. / Mutations Can Cause Large Changes in the Conformation of a Denatured Protein. In: Biochemistry. 1993 ; Vol. 32, No. 39. pp. 10359-10370.
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abstract = "Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WT SNaseA) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936–944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748–752]. Because of these and other properties of the SNaseΔ polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNaseΔ may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNaseΔ, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNaseΔ), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNaseΔ exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNaseΔ were all of the m− type (m− substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNaseΔ that were more extended and coil-like than WT SNaseΔ were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNaseΔ, as well as both the m+ and m− substituted versions of SNaseΔ, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain. The correspondence between changes in Rg produced by amino acid substitutions in SNaseΔ and the size of the denatured state predicted on the basis of solvent denaturation studies for these mutated forms supports the view that some amino acid substitutions can affect stability by changing the average conformation of the denatured state. In addition, our results are consistent with the idea that secondary structure formation and chain condensation occur simultaneously in this system.",
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Mutations Can Cause Large Changes in the Conformation of a Denatured Protein. / Flanagan, John; Kataoka, Mikio; Fujisawa, Tetsuro; Engelman, Donald M.

In: Biochemistry, Vol. 32, No. 39, 01.01.1993, p. 10359-10370.

Research output: Contribution to journalArticle

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T1 - Mutations Can Cause Large Changes in the Conformation of a Denatured Protein

AU - Flanagan, John

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N2 - Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WT SNaseA) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936–944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748–752]. Because of these and other properties of the SNaseΔ polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNaseΔ may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNaseΔ, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNaseΔ), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNaseΔ exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNaseΔ were all of the m− type (m− substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNaseΔ that were more extended and coil-like than WT SNaseΔ were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNaseΔ, as well as both the m+ and m− substituted versions of SNaseΔ, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain. The correspondence between changes in Rg produced by amino acid substitutions in SNaseΔ and the size of the denatured state predicted on the basis of solvent denaturation studies for these mutated forms supports the view that some amino acid substitutions can affect stability by changing the average conformation of the denatured state. In addition, our results are consistent with the idea that secondary structure formation and chain condensation occur simultaneously in this system.

AB - Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WT SNaseA) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936–944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748–752]. Because of these and other properties of the SNaseΔ polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNaseΔ may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNaseΔ, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNaseΔ), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNaseΔ exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNaseΔ were all of the m− type (m− substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNaseΔ that were more extended and coil-like than WT SNaseΔ were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNaseΔ, as well as both the m+ and m− substituted versions of SNaseΔ, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain. The correspondence between changes in Rg produced by amino acid substitutions in SNaseΔ and the size of the denatured state predicted on the basis of solvent denaturation studies for these mutated forms supports the view that some amino acid substitutions can affect stability by changing the average conformation of the denatured state. In addition, our results are consistent with the idea that secondary structure formation and chain condensation occur simultaneously in this system.

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