Alteration of the cystic fibrosis transmembrane conductance regulator folding pathway: Effects of the ΔF508 mutation on the thermodynamic stability and folding yield of NBD1

Bao He Qu, Philip J. Thomas

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Abstract

The cellular phenotype of the most common cystic fibrosis-causing mutation, deletion of phenylalanine 508 (ΔF508) in the amino-terminal nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), is the inability of the mutant protein to fold and transit to the apical membrane of affected epithelial cells. Expressed NBD1s were purified and folded in vitro into soluble monomers capable of binding nucleotide. Here we report that the ΔF508 mutation has little effect on the thermodynamic stability of the folded NBD1. The ΔGD 0,GdnHcl is 15.5 kJ/mol for the wild type NBD1 and 14.4 kJ/mol for NBD1ΔF. In contrast, the mutation significantly reduces the folding yield at a variety of temperatures, indicating that Phe508 makes crucial contacts during the folding process, but plays little role in stabilization of the native state. Under conditions that approximate the efficiency of maturation in vivo, the rate off-pathway is significantly increased by the disease causing mutation. These results establish a molecular mechanism for most cases of cystic fibrosis and provide insight into the complex processes by which primary sequence encodes the three-dimensional structure.

Original languageEnglish (US)
Pages (from-to)7261-7264
Number of pages4
JournalJournal of Biological Chemistry
Volume271
Issue number13
StatePublished - Mar 29 1996

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Cystic Fibrosis Transmembrane Conductance Regulator
Thermodynamics
Thermodynamic stability
Nucleotides
Cystic Fibrosis
Mutation
Mutant Proteins
Phenylalanine
Stabilization
Sequence Deletion
Monomers
Membranes
Epithelial Cells
Phenotype
Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Alteration of the cystic fibrosis transmembrane conductance regulator folding pathway: Effects of the ΔF508 mutation on the thermodynamic stability and folding yield of NBD1",
abstract = "The cellular phenotype of the most common cystic fibrosis-causing mutation, deletion of phenylalanine 508 (ΔF508) in the amino-terminal nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), is the inability of the mutant protein to fold and transit to the apical membrane of affected epithelial cells. Expressed NBD1s were purified and folded in vitro into soluble monomers capable of binding nucleotide. Here we report that the ΔF508 mutation has little effect on the thermodynamic stability of the folded NBD1. The ΔGD 0,GdnHcl is 15.5 kJ/mol for the wild type NBD1 and 14.4 kJ/mol for NBD1ΔF. In contrast, the mutation significantly reduces the folding yield at a variety of temperatures, indicating that Phe508 makes crucial contacts during the folding process, but plays little role in stabilization of the native state. Under conditions that approximate the efficiency of maturation in vivo, the rate off-pathway is significantly increased by the disease causing mutation. These results establish a molecular mechanism for most cases of cystic fibrosis and provide insight into the complex processes by which primary sequence encodes the three-dimensional structure.",
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N2 - The cellular phenotype of the most common cystic fibrosis-causing mutation, deletion of phenylalanine 508 (ΔF508) in the amino-terminal nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), is the inability of the mutant protein to fold and transit to the apical membrane of affected epithelial cells. Expressed NBD1s were purified and folded in vitro into soluble monomers capable of binding nucleotide. Here we report that the ΔF508 mutation has little effect on the thermodynamic stability of the folded NBD1. The ΔGD 0,GdnHcl is 15.5 kJ/mol for the wild type NBD1 and 14.4 kJ/mol for NBD1ΔF. In contrast, the mutation significantly reduces the folding yield at a variety of temperatures, indicating that Phe508 makes crucial contacts during the folding process, but plays little role in stabilization of the native state. Under conditions that approximate the efficiency of maturation in vivo, the rate off-pathway is significantly increased by the disease causing mutation. These results establish a molecular mechanism for most cases of cystic fibrosis and provide insight into the complex processes by which primary sequence encodes the three-dimensional structure.

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