Ligand Binding Alters the Backbone Mobility of Intestinal Fatty Acid-Binding Protein as Monitored by ¹⁵N NMR Relaxation and ¹H Exchange

Abstract

The backbone dynamics of the liganded (holo) and unliganded (apo) forms of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) have been characterized and compared using amide 15N relaxation and 1H exchange NMR measurements. The amide 1H/15N resonances for apo and holo I-FABP were assigned at 25 degrees C, and gradient- and sensitivity-enhanced 2D experiments were employed to measure l5N T1, T2, and [1H]15N NOE values and relative 1H saturation transfer rates. The 15N relaxation parameters were analyzed using five different representations of the spectral density function based on the Lipari and Szabo formalism. A majority of the residues in both apo and holo I-FABP were characterized by relatively slow hydrogen exchange rates, high generalized order parameters, and no conformational exchange terms. However, residues V26-N35, S53-R56, and A73-T76 of apo I-FABP were characterized by rapid hydrogen exchange, low order parameters, and significant conformational exchange. These residues are clustered in a single region of the protein where variability and apparent disorder were previously observed in the chemical shift analyses and in the NOE-derived NMR structures of apo I-FABP. The increased mobility and discrete disorder in the backbone of the apo protein may permit the entry of ligand into the binding cavity. We postulate that the bound fatty acid participates in a series of long-range cooperative interactions that cap and stabilize the C-terminal half of helix II and lead to an ordering of the portal region. This ligand-modulated order-disorder transition has implications for the role of I-FABP in cellular fatty acid transport and targeting.

The integrity of the α-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes

“…Intestinal and liver FABP have very similar overall conformations, as shown by the comparison of rms distance differences, the superimposition of crystallographic structural models [3], and the secondary structure assignments from their solution structures [10]. The construction of a pair of chimeric proteins by swapping the whole α helical domain between I-and LFABP demonstrated the importance of this domain in the determination of FA transfer mechanism in both proteins.…”

“…It is believed that this helical region is part of a "dynamic portal" that regulates fatty acid entry and exit from the internal cavity [10,11]. The β-barrel domain contains the ligand binding site.…”

Turn scanning by site‐directed mutagenesis: Application to the protein folding problem using the intestinal fatty acid binding protein

“…It should be noted that, in both this mutant and the N54V mutant, log&) as a function of Gdn concentration appears to be nonlinear leveling off at a rate constant close to that observed for the wild-type protein . While it is not yet clear how to interpret this result, it is of interest that the NMR data show these two regions of the protein, around N54 and G75, to be flexible (Hodsdon & Cistola, 1997a, 1997b suggesting as above that these turns are not important in the folding process. Perhaps, however, Gdn shifts the order-disorder equilibrium that occurs in these regions.…”

“…This mutation results in only slight differences in the near UV CD spectrum from wild-type (data not shown). It is of interest to note that this region of the protein is critical for ligand entry Cistola et al, 1996) and is flexible in solution (Hodsdon & Cistola, 1997a, 1997b. The small changes in stability and ligand binding and its flexibility suggests that this turn is not an important one in the folding process.…”

“…The structure of the protein has been examined both by X-ray Sacchettini & Gordon, 1993;Banaszak et al, 1994) and NMR (Hodsdon et al, 1996;Hodsdon & Cistola, 1997a, 1997bZhang et al, 1997) methods. It is an excellent model protein for folding studies, not only because of its small size and P-sheet structure, but also because it contains no cysteine or proline residues.…”