Site-directed mutagenesis was used to introduce cysteine residues into the rat intestinal fatty acid binding protein, an almost all beta-sheet protein that in the wild-type contains neither cysteine nor proline residues. Six mutants (I23C, S53C, V60C, L72C, L89C, and A104C) with a single cysteine residue substituted for a hydrophobic residue were characterized by their stability toward denaturants at pH 7.2 and 9.6, by their fluorescent properties, and by their reactivity toward the sulfhydryl modifying reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 4,4'-dipyridyl disulfide (4-PDS). In terms of protein stability, the substitutions were reasonably conservative with only two (V60C and L89C) being somewhat less stable than the wild-type. The mutant proteins differed considerably, however, in their reactivity toward the modifying reagents. One residue, Cys89, located in a hydrophobic core near a turn between two beta-strands, was unreactive, while two residues, Cys60 and Cys104, located in the middle of beta-strands in the cavity into which fatty acid binds, reacted only very slowly and were further protected by oleate. Cys53, located near a turn and partially buried, appeared to have an unusually low pK value. Two residues, Cys23 and Cys72, reacted more rapidly in the native protein than in the unfolded protein. Both residues are located near the portal for the fatty acid binding, and one, Cys72, was strongly protected from modification by the presence of oleate. Examination of the crystal structure indicates that Cys72 is not easily solvent-accessible. We conclude that this high reactivity for this residue may be a consequence of rapid conformational flexibility in this region of the structure. Study holds ProTherm entries: 4598, 4599, 4600, 4601, 4602, 4603, 4604, 4605, 4606, 4607, 4608, 4609, 4610, 4611 Extra Details: additive : EDTA(0.1 mM), all beta-sheet protein; cysteine; hydrophobic; partially buried;,conformational flexibility
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points||40|
|Proteins||Fatty acid-binding protein, brain ; Fatty acid-binding protein, intestinal|
|Assays/Quantities/Protocols||Experimental Assay: Cm buffers:Sodium pyrophosphate: 20 mM, details:Additives , pH:9.6 ; Experimental Assay: dG_H2O buffers:Sodium pyrophosphate: 20 mM, details:Additives , pH:9.6 ; Experimental Assay: Cm pH:7.2, details:Additives EDTA (0.1 mM),, buffers:Potassium phosphate: 20 mM ; Experimental Assay: dG_H2O pH:7.2, details:Additives EDTA (0.1 mM),, buffers:Potassium phosphate: 20 mM ; Derived Quantity: ddG_H2O buffers:Sodium pyrophosphate: 20 mM, details:Additives , pH:9.6 ; Derived Quantity: ddG_H2O pH:7.2, details:Additives EDTA (0.1 mM),, buffers:Potassium phosphate: 20 mM|
|Libraries||Mutations for sequence AFDGTWKVDRNENYEKFMEKMGINVVKRKLGAHDNLKLTITQEGNKFTVKESSNFRNIDVVFELGVDFAYSLADGTELTGTWTMEGNKLVGKFKRVDNGKELIAVREISGNELIQTYTYEGVEAKRIFKKE|