Trp RNA-binding attenuation protein: modifying symmetry and stability of a circular oligomer

Oliver W Bayfield; Chao-Sheng Chen; Andrea R Patterson; Weisha Luan; Callum Smits; Paul Gollnick; Alfred A Antson

doi:10.1371/journal.pone.0044309

Trp RNA-binding attenuation protein: modifying symmetry and stability of a circular oligomer

Oliver W Bayfield, Chao-Sheng Chen, Andrea R Patterson, Weisha Luan, Callum Smits, Paul Gollnick, Alfred A Antson

Chemistry

Research output: Contribution to journal › Article › peer-review

Abstract

BACKGROUND: Subunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins.

METHODOLOGY/PRINCIPAL FINDINGS: Among the wild type B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts--have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP.

CONCLUSIONS/SIGNIFICANCE: We conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion) of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology.

Original language	English
Article number	e44309
Journal	PLoS ONE
Volume	7
Issue number	9
DOIs	https://doi.org/10.1371/journal.pone.0044309
Publication status	Published - 6 Sept 2012

Keywords

Bacillus subtilis
Bacterial Proteins
Crystallography, X-Ray
Kinetics
Mass Spectrometry
Mutant Proteins
Protein Binding
Protein Stability
Protein Structure, Quaternary
Protein Structure, Secondary
Protein Subunits
RNA-Binding Proteins
Transcription Factors
Transition Temperature
Tryptophan

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10.1371/journal.pone.0044309

Cite this

@article{e0b7648706494c4f803b6bc1b5e9173c,

title = "Trp RNA-binding attenuation protein: modifying symmetry and stability of a circular oligomer",

abstract = "BACKGROUND: Subunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins.METHODOLOGY/PRINCIPAL FINDINGS: Among the wild type B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts--have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP.CONCLUSIONS/SIGNIFICANCE: We conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion) of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology.",

keywords = "Bacillus subtilis, Bacterial Proteins, Crystallography, X-Ray, Kinetics, Mass Spectrometry, Mutant Proteins, Protein Binding, Protein Stability, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Subunits, RNA-Binding Proteins, Transcription Factors, Transition Temperature, Tryptophan",

author = "Bayfield, {Oliver W} and Chao-Sheng Chen and Patterson, {Andrea R} and Weisha Luan and Callum Smits and Paul Gollnick and Antson, {Alfred A}",

year = "2012",

month = sep,

day = "6",

doi = "10.1371/journal.pone.0044309",

language = "English",

volume = "7",

journal = "PLoS ONE",

issn = "1932-6203",

publisher = "Public Library of Science",

number = "9",

}

TY - JOUR

T1 - Trp RNA-binding attenuation protein

T2 - modifying symmetry and stability of a circular oligomer

AU - Bayfield, Oliver W

AU - Chen, Chao-Sheng

AU - Patterson, Andrea R

AU - Luan, Weisha

AU - Smits, Callum

AU - Gollnick, Paul

AU - Antson, Alfred A

PY - 2012/9/6

Y1 - 2012/9/6

N2 - BACKGROUND: Subunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins.METHODOLOGY/PRINCIPAL FINDINGS: Among the wild type B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts--have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP.CONCLUSIONS/SIGNIFICANCE: We conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion) of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology.

AB - BACKGROUND: Subunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins.METHODOLOGY/PRINCIPAL FINDINGS: Among the wild type B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts--have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP.CONCLUSIONS/SIGNIFICANCE: We conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion) of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology.

KW - Bacillus subtilis

KW - Bacterial Proteins

KW - Crystallography, X-Ray

KW - Kinetics

KW - Mass Spectrometry

KW - Mutant Proteins

KW - Protein Binding

KW - Protein Stability

KW - Protein Structure, Quaternary

KW - Protein Structure, Secondary

KW - Protein Subunits

KW - RNA-Binding Proteins

KW - Transcription Factors

KW - Transition Temperature

KW - Tryptophan

U2 - 10.1371/journal.pone.0044309

DO - 10.1371/journal.pone.0044309

M3 - Article

C2 - 22970197

SN - 1932-6203

VL - 7

JO - PLoS ONE

JF - PLoS ONE

IS - 9

M1 - e44309

ER -