Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

Abstract

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty acids closely parallel their prevalences in the hepatopancreas of C. quadricarinatus as measured under specific diet conditions.

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

“…A vast number of biological applications of fluorescence anisotropy have been reported to date due to the comparable timescale of rotational diffusion of biopolymers and the fluorescence lifetime of many fluorophores. Anisotropy measurement can provide information on the shape and size of proteins and have been used to measure protein-protein associations, the fluidity of membranes, binding and conformational dynamics [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ]. However, fluorescence anisotropy is highly dependent on the environment (solvent viscosity), the size and shape of the fluorophore and the flexibility of the protein [ 2 ].…”

Discovery of the 2,4′‐Dihydroxy‐3′‐methoxypropiophenone Biosynthesis Genes in Aspergillus oryzae

“…To verify our hypothesis, we prepared recombinant PpsC protein (Figure S7A) and analyzed in vitro binding of PpsC to 3 by a competition assay using 8‐anilinonaphthalene‐1‐sulfonic acid (ANS) as an indicator according to previous reports [33,34] . First, we determined the K d value between ANS and PpsC to be 26.6±9.3 μM (Figure S7B).…”

“…To verify our hypothesis, we prepared recombinant PpsC protein ( Figure S7A) and analyzed in vitro binding of PpsC to 3 by a competition assay using 8-anilinonaphthalene-1-sulfonic acid (ANS) as an indicator according to previous reports. [33,34] First, we determined the K d value between ANS and PpsC to be 26.6 � 9.3 μM ( Figure S7B). Although the value was higher than the reported K d value (1.58 � 0.29 μM) between ANS and the epidermal fatty acid-binding protein E-FABP, [35] the affinity of ANS to PpsC was high enough to carry out displacement experiments.…”

Integrative Analysis of Proteomics and Transcriptomics of Longissimus dorsi with Different Feeding Systems in Yaks

“…It is a small-molecule protein in cells that is abundant in most tissues. FABP can participate in the transport of intracellular FAs and is vital for the uptake, transport, and metabolism of intracellular long-chain FAs [39,40]. FABP3 can transport FAs from the cytoplasmic membrane to esterification and oxidation sites, allowing them to enter the mitochondrial energy metabolism system, where FAs are oxidized and decomposed and eventually generate adenosine triphosphate (ATP).…”