Adipocyte lipid binding protein

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The adipocyte lipid binding protein (ALBP), also called as adipocyte fatty acid binding protein is a small 14.5 kDa, 10 stranded β-barrel protein found in mammalian fat cells (Ory et al 1998). ALBP is also known as 422, aP2, or p15 in the biochemical literature (Bernlohr et al., 1985; Hunt et al., 1986; Hresko et al., 1988). It belongs to a homologous family of proteins known as intracellular hydrophobic lipid binding proteins (iLBPs). ALBP is involved with fatty acid storage, trakkicing and solubilisation ( Amy et al 1999). ALBP comprises 1-3% of total soluble adipocyte protein. ALBP is one of two gene product markers used to follow the conversion of precursor to mature adipose cells. Rate of its activity is increased more than 50 fold during this conversion. The most common physiological ligand of ALBP is not certain, though it has the capacity to carry a range of fatty acids. So far from the study of protein it states that ALBP from N-terminal end, 38 residues are conserved in all ALBP species, including the first β-strand and the two α-helices that form the “lid” on the barrel. From the data so far available, the amino acid sequences of ALBP from the different species are 73% or more identical. The metabolic role of ALBP is such that when ingested lipids destined for adipose tissue are processed to eventually become a part of very low density lipoproteins. The role of ALBP is to sequester lipids from the aqueous surrounding and shuttle them back and forth from adipocyte cell membrane to the cellular organelles or to other proteins.

This essay will cover how ALBP is modified with a phenanthroline group at C117 site. The protein is registered as Phenanthroline modified Murine adipocyte lipid binding protein on PDB website (code:1A18). It's chemical and structural characterisation. How the structural techniques like X-ray crystallography are useful to determine the biological function of the protein and its impact. Also the second part of the essay will address other techniques that may be used in further understanding of the biology of the protein. The x-ray model will tell us about the chiral selectivity seen in the PHEN-ALBP. How modified version reduces the internal cavity volume, how it sterically limits the substrate interactions with reactive groups and solvent access to potential intermediates in reaction pathways.

Structural studies of Phenanthroline modified ALBP:

.Since the creation of Phenanthroline modified ALBP in 1997, several workers have studied the structure of the ALBP-PHEN with a view of understanding the biology of this protein which very little is known about. The crystal structures of various holoforms of ALBP have been solved until now and show that the fatty acid ligand bound in a large ~400A3 cavity isolated from bulk solvent. The examination of the cavity suggested being a good site for construction of an artificial catalyst. As from past studies it was clear that many ALBP structures are tolerant to modification and mutagenesis. ALBP-PHEN was crysatallised in a space group C2221, isomorphous with a mutant of ALBP. It was observed that ALBP was isomorphous with the previously solved mutant structure of ALBP, the V32D/F57H mutant at pH 6.4(ory et al,1998). This facilitated the X-ray phase determination. The previously solved mutant structures were used as starting model with its mutant residues and C117 changed to alanine. Further refinement was carried out by fitting previous mutants into electron density maps. It was then found that phenanthroline group was not easy to fit.

However, some electron density was visible for the phenanthroline group at lower σ values.

The weaker electron density suggested an approximate position for the phenanthroline ring near the portal area of ALBP residue S53. This segment is close to the suggested site of ligand entry or exit (Davis and Distafano, 1997). In the early stages of refinement, B factors for the phenanthroline group were high, reaching a maximum of 100Å2. The modification reaction seemed to be incomplete. Hence, occupancies of all the modifying atoms were set to 0.5 which resulted in a smoother b factor progression for the atoms along the phenanthroline group. The use of partial occupancy was found to be appropriate as incomplete substitution of phenanthroline group was found at C117 site. The similarity of the phenanthroline modified protein with the native ALBP was determined with the help of Least-squares method. The r.m.s.d (root mean square distance) values for native form and ALBP-PHEN were compared. From the values it was clear that both native and ALBP-PHEN have same general backbone conformation. Few changes occurred between modified and native protein. The modified protein when was compared with the crystal structure of native protein bound to fatty acids some slight differences in the conformation were observed. The majority of main chain conformational differences occurred in the portal region in helix αII and the βc and βd loop. The βc and βd loop appears to move towards the cavity slightly, whereas helix αII moves little bit away from the cavity.

The crystal structure of the phenanthroline modified ALBP attached to C117 site is shown in Fig 1. From the fig 1 we come to know that many of the atoms of the phenanthroline ring appeared disordered in the early stages of the crystal study. It was seen to be interesting that even though catalytically active form of ALBP-PHEN is bound to Cu2+ ion, the crystal structures were obtained in absence of copper. This was done because trials which included copper failed to yield crystals suitable X-ray diffraction studies. In the models obtained the rough position of the copper ion were seen to be filled by side chain oxygen atom Og, of S53. In the electron density mapping the lack of electron density for portions of phenanthroline group modification suggests that there are insufficient favourable contacts with the residues inside the cavity. Examination of the crystal structure indicated location of the modification (C117) prevents the repositioning of the phenanthroline ring on the surface of molecule without major conformational changes in the main chain. This could be supported by the result that side chain S53was found in the same position as it is in the native protein. It also showed formation of hydrogen bonds with nitrogens in the phenanthroline ring.

The research workers had examined the other potential positions for the catalytic group by maintaining the crystal conformation intact and rotating the torsinal bonds in the tether of the phenanthroline ring. The tether starts with Cα-Cβ of C117 and the torsional

bond angles are defined as CaCbSgCdCeONzCh.(ory et al, 1997). The study indicated that even a small rotation around any torsional angles in the first segment, CaCbSgCd, lead to steric conflicts. While on the other side, a rotation around CeONzof about 90⁰ show a poor contact between the oxygen atom and nearby carbon atoms in the phenanthroline ring and at the same time positions the hypothetical copper ion position near the opening found between the helix-turn-helix and the barrel itself. This study depicted that CO bond may be also perpendicular to the plane of the phenanthroline ring in the modified protein. The study was only theoretical and its orientation is shown in fig2

The crystal structures of ALBP-PHEN show residues A75, D76, F57, M20, F16, I104 and R126 which would help define enantiomer selectivity. Finally from the studies it could be summarised that it was not feasible for dynamic repositioning of the phenanthroline ring to the surface of ALBP. However, when the phenanthroline ring is rotated 90⁰ and is located in the portal region, the conformational modification appears feasible. The crystal structures also showed that ALBP-PHEN has five atoms and six torsional angles connecting the phenanthroline group to the main chain. It was assumed that the long tether for the phenanthroline modified protein accounts for the increased disorder in the catalytic moiety observed in crystal structure. The crystal structure of ALBP-PHEN does not differ significantly from the native form of protein.The electron density of the phenanthroline group appears disordered after the e -carbon atom and the electron density for the group is well ordered indicating that the phenanthroline group has a limited range of motion.


1. Structural characterization of two synthetic catalysts based on adipocyte lipid-binding protein. Ory JJ, Mazhary A, Kuang H, Davies RR, Distefano MD, Banaszak LJ.Department of Biochemistry, University of Minnesota, Minneapolis 55455, USA.

2. D.A. Bernlohr, M.A. Simpson, Adipose tissue and lipid me-tabolism, in: D.E. Vance, J. Vance (Eds.), Biochemistry of Lipids, Lipoproteins and Membranes, Elsevier, Amsterdam,1996, pp. 257^281.

3. Structural properties of the adipocyte lipid binding protein. Amy Reese-Wagoner, James Thompson, Leonard Banaszak * Biochimica et Biophysica Acta 1441 (1999) 106^116.

4. Davies,R.R. and Distefano,M.D. (1997) J. Am. Chem. Soc., 119, 11643-11652.