Utility of anion and cation combinations for phasing of protein structures.

Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Journal of Structural and Functional Genomics 05/2012; 13(3):135-43. DOI: 10.1007/s10969-012-9137-3
Source: PubMed

ABSTRACT We report the use of anionic (I(-)), cationic (Ba(2+), Cd(2+)) and ionic mixtures (I(-) plus Ba(2+)) for derivatizing liver fatty acid binding protein (LFABP) crystals. Use of cationic and anionic salts in phasing experiments revealed distinct non-overlapping sites for these ions, suggesting exclusive binding regions on LFABP. Interestingly, cations of identical charge and valency (like Ba(2+) and Cd(2+)) bound to distinct pockets on the protein surface. Furthermore, a mixture of salts containing both I(-) and Ba(2+) was very useful in phasing experiments as these oppositely charged ions bound to different regions of LFABP. Our data therefore suggest that cationic and anionic salt mixtures like BaCl(2) with NH(4)I or salts like CdI, BaI where each ion has a significant anomalous signal for a given X-ray wavelength may be valuable reagents for phasing during structure determination.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.
    Acta Crystallographica Section D Biological Crystallography 01/2014; 70(Pt 1):165-76. DOI:10.1107/S1399004713027910 · 7.23 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human liver fatty acid binding protein (hL-FABP) is the most abundant cytosolic protein in the liver. This protein plays important roles associated to partitioning of fatty acids (FAs) to specific metabolic pathways, nuclear signaling and protection against oxidative damage. The protein displays promiscuous binding properties and can bind two internal ligands, unlike FABPs from other tissues. Different topologies for the ligand located in the more accessible site have been reported, with either a 'head-in' or 'head-out' orientation of the carboxylate end. Electrospray-ionization mass spectrometry and nuclear magnetic resonance titrations are employed here in order to investigate in further detail the binding properties of this system, the equilibria established in solution and the pH dependence of the complexes. The results are consistent with two binding sites with different affinity and a unique head-out topology for the second molecule of either ligand. Competition experiments indicate a higher affinity for oleic acid relative to palmitic acid at each binding site. Copyright © 2013 John Wiley & Sons, Ltd.
    Biological Mass Spectrometry 08/2013; 48(8):895-903. DOI:10.1002/jms.3237 · 2.71 Impact Factor


Available from
May 20, 2014