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 β-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 μsec, 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.

The simplest dynamic model for an unfolded protein is a statistical coil that continually undergoes substantial conformational fluctuations. A growing number of studies indicate that the unfolded protein is not a simple random coil but rather forms transient structures. We have directly measured the rate of conformational fluctuations of unfolded intestinal fatty acid binding protein (131 aa, 15 kDa) by using fluorescence self-quenching in combination with fluorescence correlation spectroscopy. The conformational fluctuations in this state have an apparent relaxation time, τ _R , of 1.6 μsec in 3 M guanidine-HCl at pH 7 and 20°C. The value of τ _R increases with increasing solution viscosity, suggesting a diffusive process. In the molten globule state at pH 2, τ _R is 2.5 μsec, increasing further with the formation of salt-induced secondary structure. These measurements, which should be widely applicable to other systems, can provide important information about the still incompletely understood conformational properties of unfolded proteins and the mechanism of protein folding.