Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy.
Fluorescence correlation spectroscopy (FCS) measurements have been carried out on the intestinal fatty acid binding protein (IFABP) to study microsecond dynamics of the protein in its native state as well as in pH-induced intermediates. IFABP is a small (15 kDa) protein that consists mostly of antiparallel beta-strands enclosing a large central cavity into which the ligand binds. Because this protein does not contain cysteine, two cysteine mutants (Val60Cys and Phe62Cys) have been prepared and covalently modified with fluorescein. Based on fluorescence measurements, one of the mutants (Val60Flu) has the fluorescein moiety inside the cavity of the protein, whereas the fluorescein is exposed to solvent in the other (Phe62Flu). The protein modified at position 60 demonstrates the presence of a conformational event on the order of 35 microsec, which is not seen in the other mutant (Phe62Flu). The amplitude of this fast conformational event decreases sharply at low pH as the protein unfolds. Experiments measuring the diffusion as a function of pH indicate the formation of a compact state distinct from the native state at about pH 3.5. Steady state fluorescence and far-UV CD indicates that unfolding occurs at pH values below pH 3.
Chattopadhyay
Krishnananda
K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Saffarian
Saveez
S
Elson
Elliot L
EL
Frieden
Carl
C
D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004058:Diffusion; D050556:Fatty Acid-Binding Proteins; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D009363:Neoplasm Proteins; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins