RICERCA ITALIANA     

Structural studies on hydrophobic molecule-binding proteins

Università degli Studi di Verona

Abstract

X-ray crystallography, NMR and other spectroscopic techniques, mass spectrometry, non-conventional electrophoretic techniques, and amino acid sequencing will be used to characterize at the molecular level and with the highest possible degree of detail the members of a structural protein family that share the function of lipophilic ligand binding. The molecules included in this group are known to belong to a very limited number of protein folds and for two of these we have more than one protein present in the list we intend to study. Our research will also focus on the relevant role of some members of this protein family in the important function of vitamin transport in vertebrates. Our program includes two vitamin transporters: riboflavin-binding protein and human intrisic factor, transporter of vitamin B12 (cobalamin).

Principal Investigator

Ugo Luigi MONACO Università degli Studi di VERONA

Research Objectives

This program is a continuation of a research proposal that was already funded three times by this agency which allowed the participating operating units to establish sound collaborations that have produced significant results in the field. The goal of this research program was and is the application of the different and complementary techniques that are in use in the participating research units to several related aspects of the important problem of the interaction of a hydrophobic ligand with a protein evolved to bind it specifically or together with a family of structurally related ligands. The techniques available are those most prominent in modern structural analysis of biological macromolecules: X-ray crystallography, NMR, UV and visible spectroscopy, mass spectrometry, epitope mapping and other immunological methods advanced electrophoretic and conventional chemical sequencing techniques. The proteins we have selected, which were in some cases substituted as the program evolved, represent a very wide spectrum of hydrophobic binders and are actively being worked on by many important groups all over the world. We intend to study the properties of human serum albumin, which is probably the most thoroughly studied example of transport proteins. Our list includes also four lipocalins, members of a very well known structural family, alpha 1 microglobulin, beta-lactoglobulin, apolipoprotein D and prostaglandin D synthase (beta-trace). This last protein is the first lipocalin >>>

First Results

a) Albumin.
The characterization of the amniotic fluid protein post-translational modifications will be initially based on the hypothesis that the chromophore is a derivative of tryptophan catabolism. At the end of the 1st year the hypothesis will be checked and, if found to be correct, the structural properties of the adduct will be determined. If not, a detailed analysis performed using UV-vis spectrophotometry, spectrofluorimetry and CD will allow the definition of the optical properties of the adduct. In addition, the mass spectrometry analyses of the isolated tryptic fragments will have revealed the identity of the modified peptides and some properties of the bound molecule.
The molecular defects causing analbuminemia and hypoalbuminemia as well as the amino acid substitutions of the variant albumins isolated from the sera sent to us will be defined.
b) Beta-Lactoglobulin.
During the first phase we expect to produce doubly enriched (13C and 15N) porcine and bovine lactoglobulins (PLG and BLG) and to determine the 3D structures, via NMR and dynamic simulations, of PLG. At the same time we expect to have all the listed mutants and the new ones designed on the basis of experimental folding and interaction data and to determine the thermodynamic parameters relative to the unfolding of PLG
The molecular docking calculations will provide deeper knowledge on the properties of the calyx as well as on the features required for a chemical to >>>

Timescale

24 months

National and international background

Participation of the water insoluble metabolites in the biochemical reactions that take place in an aqueous environment requires a mechanism to render them water soluble and amenable to transport among different aqueous compartments. The mechanism developed by evolution is binding of these ligands to a water soluble protein. The molecules we call hydrophobic molecule-binding proteins have thus as a first and most obvious function that of rendering water soluble a hydrophobic ligand whose presence is necessary in an aqueous phase. Although solubilization and transport is the most thoroughly studied and the best understood of the functions of the lipophilic molecule-binding proteins, it is by no means the only function proposed for these macromolecules that are in many cases known to recognize other macromolecules and are believed in some cases to act as metabolic regulators.
The prototype of hydrophobic transporters and one of the proteins most intensively studied with every physicochemical technique is serum albumin (a1), the best known member of a protein family that is known to include other molecules that have not been so extensively characterized. The three-dimensional structure of human serum albumin has been determined by X-ray diffraction studies (a2, a3) and the coordinates of the model of the apoprotein as well as those of its complexes with fatty acids (a4) and warfarin (a5) are available . The study of natural mutants with substitutions in the >>>