(19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution.
The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein.
Salopek-Sondi
Branka
B
Department of Chemistry, Clarkson University, Potsdam, NY 13699-5810, USA.
Vaughan
Mark D
MD
Skeels
Matthew C
MC
Honek
John F
JF
Luck
Linda A
LA
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005461:Fluorine / Q000737:chemistry*; D008433:Mathematics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D012996:Solutions; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C488923:LivJ protein, E coli; D012996:Solutions; C028332:leucine-isoleucine-valine binding protein, bacteria; D005461:Fluorine; D014364:Tryptophan