Bound water in apo and holo bovine heart fatty‐acid‐binding protein determined by heteronuclear NMR spectroscopy

Abstract

Two-and three-dimensional heteronuclear NMR experiments have been performed to identify internally bound water molecules in the solution structure of bovine heart fatty-acid-binding protein (heart FABP). NOE and rotating-frame NOE (ROE) cross peaks between protein protons and protons of bound water molecules were observed in two-dimensional H 2O-ROE/NOE-1 H, 15 N-heteronuclear single quantum coherence spectra recorded from a uniformly 13 C/ 15 N-enriched sample of bovine heart FABP. Contacts between water protons and 23 NH protons of the protein backbone were identified. The protein structure consists of 10 antiparallel β-strands (βAϪβJ), forming two nearly orthogonal β-sheets, and a short helixturn-helix motif connecting β-strands A and B. The spatial folding resembles a β-barrel. Most of the water molecules are localized in the gap between β-strands D and E, and near the two A-helices. In the delipidated heart FABP additional contacts between water molecules and NH protons could be observed using a three-dimensional rotating frame Overhauser 1 H, 15 N heteronuclear single quantum coherence experiment obtained with a 15 N-labeled sample of apo-heart FABP.Keywords : bound water; apoprotein ; holoprotein; fatty-acid-binding protein; NMR.Fatty-acid-binding proteins (FABPs) belong to a class of cy-large cavity. Although the conformations of bound ligands differ tosolic, non-enzymic proteins with high non-covalent affinity for in the distinct FABP types, the conformation of the apo form of long-chain fatty acids and molecular masses of 14Ϫ15 kDa [1, the protein is very similar to the holo form [14]. 2]. FABPs have been isolated from a number of fatty-acid-me-A solution structure of bovine heart FABP has been pretabolizing tissues of mammals, fish, chicken, and insect [2]. It sented previously [15Ϫ17]. The fatty acid ligand is bound in a is proposed that fatty-acid-binding proteins serve as a pool for U-shaped conformation, within the binding cavity [17]. The solubilized fatty acids and protect the cells from the detergent crystal structure of the homologous heart FABP of human museffects of high fatty acid concentrations [3]. They are involved cle with bound fatty acid revealed 13 water molecules within in the cellular uptake of fatty acids and in the intracellular trans-this cavity. Two of these water molecules interact with the carport of fatty acids to the mitochondria. They may modulate en-boxylate group of the fatty acid. A mechanism for fatty acid zyme activities and have an influence on cell growth and cell binding is proposed by Young and coworkers [5] including the differentiation [2]. Nevertheless the precise physiological func-rearrangement of some sidechains and water molecules. Calculation of FABP still remains to be elucidated.tions of the solvent-accessible surface of human muscle heart At present seven distinct FABP types are known which have FABP suggest that the internal cavity is connected to the exterbeen isolated from liver, intestine, heart, adipocyte, myelin-type nal solvent by a portal area w...

A “structural” water molecule in the family of fatty acid binding proteins

“…Analysis of water diffusion in MD simulations suggests that most water molecules are in a motional regime close to the limit at which protein-water nuclear Overhauser effects are expected to vanish~although this results is for apo I-FABP!. Intriguingly, based on similar NMR experiments, Mesgarzadeh et al 1998! suggested 23 contacts between long-lived, internally bound water molecules in holo bovine H-FABP.…”

“…have been recently reported Mesgarzadeh et al, 1998!. These authors reported long-lived hydration water to be in contact with NH groups of Val84, Val68, and Gly67 in both apo and holo H-FABP, but they did not comment specifically on the position of water molecules which could give rise to these contacts.…”

Dynamics of internal water in fatty acid binding protein: Computer simulations and comparison with experiments

“…17 Cross-relaxation studies of I-FABP hydration were recently completed in our laboratory, 18 and similar studies of related bovine heart fatty acid binding protein (H-FABP) have also been described. 19 In this work, we report three independent one-nanosecond MD simulations of fully solvated apo I-FABP. We analyze the instantaneous number of internal water molecules as a function of the simulation time, evaluate potential pathways for the exchange of internal water molecules, and estimate their mean-square fluctuations.…”

Water‐protein interactions in the molten‐globule state of carbonic anhydrase b: An NMR spin‐diffusion study

“…For more than a decade, many researchers have undertaken the challenging task of investigating the mechanisms of interaction between water molecules and globular proteins~Ooi & Oobatake, 1988;Otting et al, 1991aOtting et al, , 1991bHolak et al, 1992;Levitt & Park, 1993;Willams et al, 1994;Alonso & Daggett, 1995;Denisov et al, 1995Denisov et al, , 1999Ernst et al, 1995;Bhattacharjya & Balaram, 1997;Takano et al, 1997;Mesgarzadeh et al, 1998;Shortle, 1999!. Some of these authors have examined the possible role of water as a stabilizing~or destabilizing!…”

“…Nevertheless, the introduction of multiparametric methods, which are capable of monitoring several different properties during the same experiment, may represent an important step forward in the study of such protein conformations. Multidimensional NMR can provide significant information about where the interaction between water and protein molecules takes place at the protein surface, the quantities of water molecules and protein cavities and lifetimes of the water molecules, among other dynamic characteristics~Willams et Alonso & Daggett, 1995;Denisov et al, 1995;Ernst et al, 1995;Otting, 1997;Mesgarzadeh et al, 1998!. NMR allows us to see almost all the spectral compounds in the sample and to measure nuclear Overhauser effects~NOE!, relaxation times, spinspin couplings, and so on.…”