Intestinal fatty acid binding protein: folding of fluorescein-modified proteins

Abstract

The rat intestinal fatty acid binding protein is an almost all beta-sheet protein that encloses a large interior cavity into which the fatty acid ligand binds. The protein contains neither cysteine nor proline. In a previous report, six site-directed mutants were obtained, each having a single cysteine residue [Jiang, N., & Frieden, C., (1993) Biochemistry 32, 11015-11021] either in a turn or pointed into the cavity. In this report, each mutant has been unfolded in denaturant and modified with 5-iodoacetamido-fluorescein to introduce a large, bulky, and fluorescent group into the protein at a known position. In all cases, fluorescence changes indicated that the modified protein refolded, and circular dichroism measurements suggested that the refolded protein appeared to be mostly beta-sheet. Denaturation curves suggest that for two mutants intermediate structures exist at denaturant concentrations well below the midpoint of the unfolding curve. For each modified, folded protein, one- and two-dimensional 1H NMR spectra were accumulated and compared to the unmodified and wild-type proteins. While the spectra for the modified proteins showed a number of changes in chemical shifts, they were also consistent with folded proteins on the basis of the degree of chemical shift dispersion. Of the six modified mutant proteins, two appear to have the fluorescein group located in the cavity, but only one of these did not bind fatty acid. The remaining modified proteins are capable of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy

“…The stability of both the mutants is similar with identical mid-points (Table 1). It can be seen that a large fluorescence change occurs with the V60Flu protein on unfolding which indicates that the fluorescein is inside the cavity consistent with earlier NMR data using V60Flu (13). The different behavior for F62Flu (Fig.…”

“…Equilibrium unfolding experiments using different cysteine modified mutants of IFABP have been studied earlier (12,13). Unfolding of mutant proteins with fluorescein attached to positions 60, 89, and 104 was accompanied by a significant increase in fluorescence intensity at 517 nm (excitation wavelength of 490 nm).…”

“…4 assumes (i) an inter-conversion of two states of the protein with a chemical relaxation time, R , and (ii) that the fluorescence of one state is significantly quenched compared with the other. We have shown elsewhere that fluorescein fluorescence is quenched in the native state of V60Flu as a result of being inside the cavity (13). It is important, however, to understand the quenching of fluorescein in the native protein to gain further insight about what the microsecond dynamic measurement represents.…”

“…The reaction with fluorescein or with Alexa488Val60Cys was carried out by using a published procedure (13). For acid unfolding experiments, a series of solutions of different pH values were prepared by using 2 mM citrate buffer.…”

“…In earlier studies, we mutated specific residues of IFABP to cysteine and examined the properties of the mutated proteins (12) as well as proteins for which the fluorescent probe fluorescein was covalently attached to the cysteine residue (13). These studies showed that the probe attached to cysteine at position 60 (V60C) was located within the interior cavity and that the fluorescence properties of the probe were sensitive to the unfolding transition.…”