The amino-terminal region of the long-chain fatty acid transport protein FadL contains an externally exposed domain required for bacteriophage T2 binding

Gaetano Cristalli, Concetta C DiRusso, Paul N Black

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Abstract

The fatty acid transport protein FadL from Escherichia coli is predicted to be rich in β-structure and span the outer membrane multiple times to form a long-chain fatty acid specific channel. Proteolysis of FadL within whole cells, total membranes, and isolated outer membranes identified two trypsin- sensitive sites, both predicted to be in externally exposed loops of FadL. Amino acid sequence analysis of the proteolytic fragments determined that the first followed R93 and yielded a peptide beginning with 94S-L-K-A-D-N-I- A-P-T-A104 while the second followed R384 and yielded a peptide beginning with 385S-I-S-I-P-D-Q-D-R-F-W395. Proteolysis using trypsin eliminated the bacteriophage T2 binding activity associated with FadL, suggesting the T2 binding domain within FadL requires elements within one of these extracellular loops. A peptide corresponding to the amino-terminal region of FadL (FadL(28-160)) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the amino, proximal extracellular loop of the protein confers T2 binding activity. Using an artificial neural network (NN) topology prediction method in combination with Gibbs motif sampling, a predicted topology of FadL within the outer membrane was developed. According to this model, FadL spans the outer membrane 20 times as antiparallel β-strands. The 20 antiparallel β-strands are presumed to form a β-barrel specific for long-chain fatty acids. On the basis of our previous studies evaluating the function of FadL using site-specific mutagenesis of the fadL gene, proteolysis of FadL within outer membranes, and studies using the FadL(28- 160) peptide, the predicted extracellular regions between β-strands 1 and 2 and β-strands 3 and 4 are expected to contribute to a domain of the protein required for long-chain fatty acid and bacteriophage T2 binding. The first trypsin-sensitive site (R93) lies between predicted β-strands 3 and 4 while the second (R384) is between β-strands 17 and 18. The trypsin- resistant region of FadL is predicted to contain 13 antiparallel β-strands and contribute to the long-chain fatty acid specific channel. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)324-333
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume377
Issue number2
DOIs
StatePublished - May 15 2000

Fingerprint

Fatty Acid Transport Proteins
Bacteriophage T4
Bacteriophages
Trypsin
Proteolysis
Membranes
Fatty Acids
Peptides
Topology
Protein Sequence Analysis
Mutagenesis
Site-Directed Mutagenesis
Escherichia coli
Cell Membrane
Proteins
Genes
Sampling
Neural networks
Amino Acids

Keywords

  • Long-chain fatty acid
  • Membrane
  • Protein
  • Transport

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

@article{64e42566af914066bc9e06cc34acd3eb,
title = "The amino-terminal region of the long-chain fatty acid transport protein FadL contains an externally exposed domain required for bacteriophage T2 binding",
abstract = "The fatty acid transport protein FadL from Escherichia coli is predicted to be rich in β-structure and span the outer membrane multiple times to form a long-chain fatty acid specific channel. Proteolysis of FadL within whole cells, total membranes, and isolated outer membranes identified two trypsin- sensitive sites, both predicted to be in externally exposed loops of FadL. Amino acid sequence analysis of the proteolytic fragments determined that the first followed R93 and yielded a peptide beginning with 94S-L-K-A-D-N-I- A-P-T-A104 while the second followed R384 and yielded a peptide beginning with 385S-I-S-I-P-D-Q-D-R-F-W395. Proteolysis using trypsin eliminated the bacteriophage T2 binding activity associated with FadL, suggesting the T2 binding domain within FadL requires elements within one of these extracellular loops. A peptide corresponding to the amino-terminal region of FadL (FadL(28-160)) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the amino, proximal extracellular loop of the protein confers T2 binding activity. Using an artificial neural network (NN) topology prediction method in combination with Gibbs motif sampling, a predicted topology of FadL within the outer membrane was developed. According to this model, FadL spans the outer membrane 20 times as antiparallel β-strands. The 20 antiparallel β-strands are presumed to form a β-barrel specific for long-chain fatty acids. On the basis of our previous studies evaluating the function of FadL using site-specific mutagenesis of the fadL gene, proteolysis of FadL within outer membranes, and studies using the FadL(28- 160) peptide, the predicted extracellular regions between β-strands 1 and 2 and β-strands 3 and 4 are expected to contribute to a domain of the protein required for long-chain fatty acid and bacteriophage T2 binding. The first trypsin-sensitive site (R93) lies between predicted β-strands 3 and 4 while the second (R384) is between β-strands 17 and 18. The trypsin- resistant region of FadL is predicted to contain 13 antiparallel β-strands and contribute to the long-chain fatty acid specific channel. (C) 2000 Academic Press.",
keywords = "Long-chain fatty acid, Membrane, Protein, Transport",
author = "Gaetano Cristalli and DiRusso, {Concetta C} and Black, {Paul N}",
year = "2000",
month = "5",
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volume = "377",
pages = "324--333",
journal = "Archives of Biochemistry and Biophysics",
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TY - JOUR

T1 - The amino-terminal region of the long-chain fatty acid transport protein FadL contains an externally exposed domain required for bacteriophage T2 binding

AU - Cristalli, Gaetano

AU - DiRusso, Concetta C

AU - Black, Paul N

PY - 2000/5/15

Y1 - 2000/5/15

N2 - The fatty acid transport protein FadL from Escherichia coli is predicted to be rich in β-structure and span the outer membrane multiple times to form a long-chain fatty acid specific channel. Proteolysis of FadL within whole cells, total membranes, and isolated outer membranes identified two trypsin- sensitive sites, both predicted to be in externally exposed loops of FadL. Amino acid sequence analysis of the proteolytic fragments determined that the first followed R93 and yielded a peptide beginning with 94S-L-K-A-D-N-I- A-P-T-A104 while the second followed R384 and yielded a peptide beginning with 385S-I-S-I-P-D-Q-D-R-F-W395. Proteolysis using trypsin eliminated the bacteriophage T2 binding activity associated with FadL, suggesting the T2 binding domain within FadL requires elements within one of these extracellular loops. A peptide corresponding to the amino-terminal region of FadL (FadL(28-160)) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the amino, proximal extracellular loop of the protein confers T2 binding activity. Using an artificial neural network (NN) topology prediction method in combination with Gibbs motif sampling, a predicted topology of FadL within the outer membrane was developed. According to this model, FadL spans the outer membrane 20 times as antiparallel β-strands. The 20 antiparallel β-strands are presumed to form a β-barrel specific for long-chain fatty acids. On the basis of our previous studies evaluating the function of FadL using site-specific mutagenesis of the fadL gene, proteolysis of FadL within outer membranes, and studies using the FadL(28- 160) peptide, the predicted extracellular regions between β-strands 1 and 2 and β-strands 3 and 4 are expected to contribute to a domain of the protein required for long-chain fatty acid and bacteriophage T2 binding. The first trypsin-sensitive site (R93) lies between predicted β-strands 3 and 4 while the second (R384) is between β-strands 17 and 18. The trypsin- resistant region of FadL is predicted to contain 13 antiparallel β-strands and contribute to the long-chain fatty acid specific channel. (C) 2000 Academic Press.

AB - The fatty acid transport protein FadL from Escherichia coli is predicted to be rich in β-structure and span the outer membrane multiple times to form a long-chain fatty acid specific channel. Proteolysis of FadL within whole cells, total membranes, and isolated outer membranes identified two trypsin- sensitive sites, both predicted to be in externally exposed loops of FadL. Amino acid sequence analysis of the proteolytic fragments determined that the first followed R93 and yielded a peptide beginning with 94S-L-K-A-D-N-I- A-P-T-A104 while the second followed R384 and yielded a peptide beginning with 385S-I-S-I-P-D-Q-D-R-F-W395. Proteolysis using trypsin eliminated the bacteriophage T2 binding activity associated with FadL, suggesting the T2 binding domain within FadL requires elements within one of these extracellular loops. A peptide corresponding to the amino-terminal region of FadL (FadL(28-160)) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the amino, proximal extracellular loop of the protein confers T2 binding activity. Using an artificial neural network (NN) topology prediction method in combination with Gibbs motif sampling, a predicted topology of FadL within the outer membrane was developed. According to this model, FadL spans the outer membrane 20 times as antiparallel β-strands. The 20 antiparallel β-strands are presumed to form a β-barrel specific for long-chain fatty acids. On the basis of our previous studies evaluating the function of FadL using site-specific mutagenesis of the fadL gene, proteolysis of FadL within outer membranes, and studies using the FadL(28- 160) peptide, the predicted extracellular regions between β-strands 1 and 2 and β-strands 3 and 4 are expected to contribute to a domain of the protein required for long-chain fatty acid and bacteriophage T2 binding. The first trypsin-sensitive site (R93) lies between predicted β-strands 3 and 4 while the second (R384) is between β-strands 17 and 18. The trypsin- resistant region of FadL is predicted to contain 13 antiparallel β-strands and contribute to the long-chain fatty acid specific channel. (C) 2000 Academic Press.

KW - Long-chain fatty acid

KW - Membrane

KW - Protein

KW - Transport

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U2 - 10.1006/abbi.2000.1794

DO - 10.1006/abbi.2000.1794

M3 - Article

VL - 377

SP - 324

EP - 333

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

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