Kinetic and thermodynamic analysis of Bradyrhizobium japonicum PutA-membrane associations

Weimin Zhang, Navasona Krishnan, Donald F Becker

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

In Escherichia coli, proline induces tight membrane binding of the PutA flavoenzyme and transforms PutA from a transcriptional repressor to a membrane-associated proline catabolic enzyme. In other gram-negative bacteria such as Bradyrhizobium japonicum, PutA lacks DNA binding activity and functions only as a proline catabolic enzyme. Here, we characterize the membrane binding properties of PutA from B. japonicum (BjPutA) to address whether proline regulates BjPutA-lipid binding similar to Escherichia coli PutA (EcPutA). Surface plasmon resonance (SPR) kinetic measurements of BjPutA-lipid binding show BjPutA forms a complex with lipids in the absence and presence of proline with similar dissociation constant (KD) values of 2.5 and 1.7 nM, respectively. SPR experiments using differently charged lipid bilayers indicate BjPutA selectively binds negatively charged lipids, which contrasts with the charge independent membrane binding of EcPutA. Analysis of BjPutA-lipid binding by isothermal titration calorimetry at 25°C revealed an endothermic binding reaction that is entropically driven. This work shows that BjPutA-membrane associations vary significantly from EcPutA.

Original languageEnglish (US)
Pages (from-to)174-183
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume445
Issue number1
DOIs
StatePublished - Jan 1 2006

Fingerprint

Bradyrhizobium
Thermodynamics
Proline
Escherichia coli
Membranes
Lipids
Kinetics
Surface Plasmon Resonance
Surface plasmon resonance
Calorimetry
Lipid bilayers
Lipid Bilayers
Enzymes
Gram-Negative Bacteria
Titration
Bacteria
DNA
Experiments

Keywords

  • Flavoprotein
  • Multifunctional enzyme
  • Protein membrane associations
  • Surface plasmon resonance

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Kinetic and thermodynamic analysis of Bradyrhizobium japonicum PutA-membrane associations. / Zhang, Weimin; Krishnan, Navasona; Becker, Donald F.

In: Archives of Biochemistry and Biophysics, Vol. 445, No. 1, 01.01.2006, p. 174-183.

Research output: Contribution to journalArticle

@article{9b361ef959544157aab6913c635827c6,
title = "Kinetic and thermodynamic analysis of Bradyrhizobium japonicum PutA-membrane associations",
abstract = "In Escherichia coli, proline induces tight membrane binding of the PutA flavoenzyme and transforms PutA from a transcriptional repressor to a membrane-associated proline catabolic enzyme. In other gram-negative bacteria such as Bradyrhizobium japonicum, PutA lacks DNA binding activity and functions only as a proline catabolic enzyme. Here, we characterize the membrane binding properties of PutA from B. japonicum (BjPutA) to address whether proline regulates BjPutA-lipid binding similar to Escherichia coli PutA (EcPutA). Surface plasmon resonance (SPR) kinetic measurements of BjPutA-lipid binding show BjPutA forms a complex with lipids in the absence and presence of proline with similar dissociation constant (KD) values of 2.5 and 1.7 nM, respectively. SPR experiments using differently charged lipid bilayers indicate BjPutA selectively binds negatively charged lipids, which contrasts with the charge independent membrane binding of EcPutA. Analysis of BjPutA-lipid binding by isothermal titration calorimetry at 25°C revealed an endothermic binding reaction that is entropically driven. This work shows that BjPutA-membrane associations vary significantly from EcPutA.",
keywords = "Flavoprotein, Multifunctional enzyme, Protein membrane associations, Surface plasmon resonance",
author = "Weimin Zhang and Navasona Krishnan and Becker, {Donald F}",
year = "2006",
month = "1",
day = "1",
doi = "10.1016/j.abb.2005.10.022",
language = "English (US)",
volume = "445",
pages = "174--183",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Kinetic and thermodynamic analysis of Bradyrhizobium japonicum PutA-membrane associations

AU - Zhang, Weimin

AU - Krishnan, Navasona

AU - Becker, Donald F

PY - 2006/1/1

Y1 - 2006/1/1

N2 - In Escherichia coli, proline induces tight membrane binding of the PutA flavoenzyme and transforms PutA from a transcriptional repressor to a membrane-associated proline catabolic enzyme. In other gram-negative bacteria such as Bradyrhizobium japonicum, PutA lacks DNA binding activity and functions only as a proline catabolic enzyme. Here, we characterize the membrane binding properties of PutA from B. japonicum (BjPutA) to address whether proline regulates BjPutA-lipid binding similar to Escherichia coli PutA (EcPutA). Surface plasmon resonance (SPR) kinetic measurements of BjPutA-lipid binding show BjPutA forms a complex with lipids in the absence and presence of proline with similar dissociation constant (KD) values of 2.5 and 1.7 nM, respectively. SPR experiments using differently charged lipid bilayers indicate BjPutA selectively binds negatively charged lipids, which contrasts with the charge independent membrane binding of EcPutA. Analysis of BjPutA-lipid binding by isothermal titration calorimetry at 25°C revealed an endothermic binding reaction that is entropically driven. This work shows that BjPutA-membrane associations vary significantly from EcPutA.

AB - In Escherichia coli, proline induces tight membrane binding of the PutA flavoenzyme and transforms PutA from a transcriptional repressor to a membrane-associated proline catabolic enzyme. In other gram-negative bacteria such as Bradyrhizobium japonicum, PutA lacks DNA binding activity and functions only as a proline catabolic enzyme. Here, we characterize the membrane binding properties of PutA from B. japonicum (BjPutA) to address whether proline regulates BjPutA-lipid binding similar to Escherichia coli PutA (EcPutA). Surface plasmon resonance (SPR) kinetic measurements of BjPutA-lipid binding show BjPutA forms a complex with lipids in the absence and presence of proline with similar dissociation constant (KD) values of 2.5 and 1.7 nM, respectively. SPR experiments using differently charged lipid bilayers indicate BjPutA selectively binds negatively charged lipids, which contrasts with the charge independent membrane binding of EcPutA. Analysis of BjPutA-lipid binding by isothermal titration calorimetry at 25°C revealed an endothermic binding reaction that is entropically driven. This work shows that BjPutA-membrane associations vary significantly from EcPutA.

KW - Flavoprotein

KW - Multifunctional enzyme

KW - Protein membrane associations

KW - Surface plasmon resonance

UR - http://www.scopus.com/inward/record.url?scp=29244452111&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=29244452111&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2005.10.022

DO - 10.1016/j.abb.2005.10.022

M3 - Article

VL - 445

SP - 174

EP - 183

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

ER -