Intestinal fatty acid binding protein: characterization of mutant proteins containing inserted cysteine residues.


Abstract

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

Submission Details

ID: WH7x25aa

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Jiang N;Frieden C,Biochemistry (1993) Intestinal fatty acid binding protein: characterization of mutant proteins containing inserted cysteine residues. PMID:8218166
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1URE 1997-03-12 NMR STRUCTURE OF INTESTINAL FATTY ACID-BINDING PROTEIN COMPLEXED WITH PALMITATE, 20 STRUCTURES
1AEL 1997-04-01 NMR STRUCTURE OF APO INTESTINAL FATTY ACID-BINDING PROTEIN, 20 STRUCTURES
1SA8 2004-06-08 THE NMR STRUCTURE OF A STABLE AND COMPACT ALL-beta-SHEET VARIANT OF INTESTINAL FATTY ACID-BINDING PROTEIN
1A57 1998-05-27 THE THREE-DIMENSIONAL STRUCTURE OF A HELIX-LESS VARIANT OF INTESTINAL FATTY ACID BINDING PROTEIN, NMR, 20 STRUCTURES
1T8V 2005-10-04 The NMR structure of d34a i-fabp: implications for the determinants of ligand binding stoichiometry
1IFC 1994-01-31 1.19 REFINEMENT OF THE STRUCTURE OF RECOMBINANT RAT INTESTINAL FATTY ACID-BINDING APOPROTEIN AT 1.2 ANGSTROMS RESOLUTION
1ICM 1994-01-31 1.5 ESCHERICHIA COLI-DERIVED RAT INTESTINAL FATTY ACID BINDING PROTEIN WITH BOUND MYRISTATE AT 1.5 A RESOLUTION AND I-FABPARG106-->GLN WITH BOUND OLEATE AT 1.74 A RESOLUTION
3AKN 2011-07-20 1.6 X-ray structure of iFABP from human and rat with bound fluorescent fatty acid analogue
1ICN 1994-01-31 1.74 ESCHERICHIA COLI-DERIVED RAT INTESTINAL FATTY ACID BINDING PROTEIN WITH BOUND MYRISTATE AT 1.5 A RESOLUTION AND I-FABPARG106-->GLN WITH BOUND OLEATE AT 1.74 A RESOLUTION
1IFB 1992-01-15 1.96 REFINED APOPROTEIN STRUCTURE OF RAT INTESTINAL FATTY ACID BINDING PROTEIN PRODUCED IN ESCHERICHIA COLI
2IFB 1992-01-15 2.0 CRYSTAL STRUCTURE OF RAT INTESTINAL FATTY-ACID-BINDING PROTEIN. REFINEMENT AND ANALYSIS OF THE ESCHERICHIA COLI-DRIVED PROTEIN WITH BOUND PALMITATE
1DC9 2000-03-20 2.1 PROPERTIES AND CRYSTAL STRUCTURE OF A BETA-BARREL FOLDING MUTANT, V60N INTESTINAL FATTY ACID BINDING PROTEIN (IFABP)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.0 Fatty acid-binding protein, brain P51880 FABP7_MOUSE
100.0 Fatty acid-binding protein, brain P55051 FABP7_RAT
92.4 Fatty acid-binding protein, intestinal P55050 FABPI_MOUSE
100.0 Fatty acid-binding protein, intestinal P02693 FABPI_RAT