A Functional R Domain from Cystic Fibrosis Transmembrane Conductance Regulator Is Predominantly Unstructured in Solution

Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708-831 of CFTR. Phosphorylate...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2000-05, Vol.97 (10), p.5657-5662
Main Authors: Ostedgaard, Lynda S., Baldursson, Olafur, Vermeer, Daniel W., Welsh, Michael J., Robertson, Andrew D.
Format: Article
Language:English
Subjects:
Amino acids
Anatomy & physiology
Biochemistry
Biological Sciences
Circular Dichroism
Cloning, Molecular
Cyclic AMP-Dependent Protein Kinases - metabolism
Cystic fibrosis
Cystic Fibrosis Transmembrane Conductance Regulator - chemistry
Cystic Fibrosis Transmembrane Conductance Regulator - genetics
Cystic Fibrosis Transmembrane Conductance Regulator - physiology
Escherichia coli
Exons
Humans
Imidazoles
Inclusion bodies
Mathematical functions
Medical research
Models, Molecular
Mutagenesis, Site-Directed
Phosphorylation
Protein Structure, Secondary
Proteins
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Sequence Deletion
Sequencing
Solutions
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Summary:Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708-831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.100588797