Charge modification at conserved positively charged residues of fatty acid binding protein (FABP) from the giant liver fluke Fasciola gigantica: its effect on oligomerization and binding properties.
Fatty acid binding proteins (FABPs) are capable of binding hydrophobic ligands with high affinity; thereby facilitating the cellular uptake and intracellular trafficking of fatty acids. In this study, functional characteristics of a cytoplasmic FABP from the giant liver fluke Fasciola gigantica (FgFABP) were determined. Binding of a fluorescent fatty acid analogue 11-[[5-dimethy aminonaphtalene-1-sulphonyl] amino] undecanoic acid (DAUDA) to FgFABP resulted in changes in the emission spectrum. The optimal excitation wavelength and maximum emission of fluorescence for binding activities with DAUDA were 350 nm and 550 nm, respectively. The binding activity for DAUDA was determined from titration experiments and revealed a Kd value of 2.95+/-0.54 microM. Furthermore, we found that cross-linking profile of FgFABP with dithiobis-(succinimidylpropionate) (DSP) in the presence of DAUDA resulted in increased formation of higher-ordered oligomers compared to that in the absence of DAUDA. We also replaced five highly conserved positively charged residues (K9, K58, K91, R107 and K131) with alanine and studied their oligomerization and binding properties of the modified FgFABPs. The obtained data demonstrate that these residues do not appear to be involved in oligomerization. However, the K58A and R107A substitutions exhibited a reduction in binding affinities. K91A and R107A revealed an increase in maximal specific binding.
Janvilisri
Tavan
T
Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand. sctcv@mahidol.ac.th
Likitponrak
Wichai
W
Chunchob
Supatra
S
Grams
Rudi
R
Vichasri-Grams
Suksiri
S
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D019281:Dimerization; D005210:Fasciola hepatica / Q000737:chemistry / Q000235:genetics; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D050556:Fatty Acid-Binding Proteins