Lucia Zetta

Monomeric bovine β-lactoglobulin adopts a β-barrel fold at pH 2

We have determined a crude structure of the apo form of bovine β‐lactoglobulin, a protein of 162 amino acid residues with a molecular mass of 18 kDa, at a low pH on the basis of data collected using only homonuclear 1H NMR spectroscopy. An ensemble of protein conformations was calculated with the distance‐geometry algorithm for NMR applications (DYANA). The monomeric protein at low pH adopts a β‐barrel fold, well‐superimposable on the structure determined by X‐ray crystallography for the dimer at physiological pH. NMR evidence suggests the presence of disordered loop regions and terminal segments. Structural differences between the monomer at pH 2 and the dimer at pH 7, obtained by X‐ray crystallography, are discussed, paying particular attention to surface electrostatic properties, in view of the high charge state of the protein at low pH.

Partially folded structure of monomeric bovine β-lactoglobulin

Bovine β‐LG (β‐lactoglobulin) has been studied under a variety of solution conditions by one‐ and two‐dimensional NMR spectroscopy. At highly acidic pH (pH = 2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured β‐sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of β‐LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.

Identification of a conserved hydrophobic cluster in partially folded bovine β-lactoglobulin at pH 2

BACKGROUNDnNMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions formed during folding. Although the complete structural characterization of partially folded proteins is a very difficult task, the identification of structured subsets, such as hydrophobic clusters, is of value in understanding the structural organization of such states. Here, we report the NMR characterization, in acidic conditions (pH 2), of a well-defined hydrophobic cluster localized in the core of bovine beta-lactoglobulin.nnnRESULTSnThe existence of a small hydrophobic cluster present in the lipocalin protein family has been assessed on the basis of structural alignment and NRM data obtained for the partially folded bovine beta-lactoglobulin. The presence of the cluster had been predicted identifying those residues that are highly conserved in most members of the family. An NMR study conducted at pH 2, where the protein exhibits a very stable beta-core together with disordered regions, reveals the presence of NOEs among sidechains of 11 hydrophobic residues centered around Trp19 and pointing towards the interior of the protein. This buried cluster is found to be unusually stable at pH 2, not only at room temperature but also at 323K. Furthermore, conserved hydrophobic residues pointing towards the surface of the protein define a hydrophobic surface patch located in a groove between the strands and the helix.nnnCONCLUSIONSnThe detected buried cluster most likely plays an important role in bovine beta-lactoglobulin stability. The analysis of five structurally related proteins reveals that the same extended cluster is present in these structures. We propose that the buried cluster may represent the internal binding site as well and that the hydrophobic surface patch is involved in a second external binding site.

NMR-based modeling and binding studies of a ternary complex between chicken liver bile acid binding protein and bile acids.

Chicken liver bile acid binding protein (cL‐BABP) is involved in bile acid transport in the liver cytosol. A detailed study of the mechanism of binding and selectivity of bile acids binding proteins towards the physiological pool of bile salts is a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. In the present study, we modeled the ternary complex of cL‐BABP with two molecules of bile salts using the data driven docking program HADDOCK on the basis of NMR and mass spectrometry data. Docking resulted in good 3D models, satisfying the majority of experimental restraints. The docking procedure represents a necessary step to help in the structure determination and in functional analysis of such systems, in view of the high complexity of the 3D structure determination of a ternary complex with two identical ligands. HADDOCK models show that residues involved in binding are mainly located in the C‐terminal end of the protein, with two loops, CD and EF, playing a major role in ligand binding. A spine, comprising polarresidues pointing toward the protein interior and involved in motion communication, has a prominent role in ligand interaction. The modeling approach has been complemented with NMR interaction and competition studies of cL‐BABP with chenodeoxycholic and cholic acids. A higher affinity for chenodeoxycholic acid was observed and a Kd upper limit estimate was obtained. The binding is highly cooperative and no site selectivity was detected for the different bile salts, thus indicating that site selectivity and cooperativity are not correlated. Differences in physiological pathways and bile salt pools in different species is discussed in light of the binding results thus enlarging the body of knowledge of BABPs biological functions. Proteins 2007.

Equilibrium unfolding CD studies of bovine beta-lactoglobulin and its 14-52 fragment at acidic pH.

Bovine beta-lactoglobulin represents an interesting example of context-dependent secondary structure induction. In fact, secondary structure predictions indicated that this beta-barrel protein has a surprisingly high alpha-helical preference, which was retained for short fragments. Cooperative transitions from the native beta-sheet to alpha-helical structures were additionally induced by organic solvents, in particular trifluoroethanol. As a result of this high alpha-helical preference, it has been proposed that non-native alpha-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better understanding of the processes that underlie conformational plasticity in this protein, CD measurements in the presence of increasing amounts of urea and in the presence of organic solvents were performed. Urea unfolding studies, performed at pH 2.1 and 37 degrees C, revealed an apparent two-state transition, and afforded no evidence of non native alpha-helical intermediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with higher molar concentration urea, lead to partial misfolding. A 29-mer peptide covering the region of strands a and b of the intact protein, characterized by the presence of 4/3 heptad repeats, was synthesized and studied by CD in the presence of different solvents. On the basis of the obtained results, a mechanism was proposed to explain the structural transition from the beta to alpha structure, provoked by organic solvents in the intact protein.

Electrospray Mass Spectrometry: Complexation Between 1-Anilinonaphthalene-8-sulphonate and Proteins

Complexation of the hydrophobic fluorescent dye 1-anilinonaphthalene-8-sulphonate with a number of proteins was examined by electrospray ionization mass spectrometry (ESMS). The apparent agreement between the present data and those obtained by fluorescence spectroscopy underlines the role of ESMS in the overall approach to protein folding and unfolding.

CD AND NMR STRUCTURAL CHARACTERIZATION OF CERATOTOXINS, NATURAL PEPTIDES WITH ANTIMICROBIAL ACTIVITY

Antibacterial properties of the secretion from the female reproductive accessory glands of medfly Ceratitis capitata are mostly ascribed to the presence of two peptides, ceratotoxin A and B, which exhibit a strong activity against gram-positive and gram-negative bacterial strains, and show sequence and function homology with cecropins, melittin, and magainins. CD experiments performed in different solvents indicate the presence of a significant content of helical structures in organic solvent. Two-dimensional nmr results for ceratotoxin A in methanol show a helical behavior for the 8-25 region of the peptide. A ramachandran classification of each residue for the structures obtained from distance geometry calculations lead to the definition of four structural families in which the central segment 10-19 is always helical and differences refer to residues 8-9 and 19-23. A sequence analysis of the two ceratotoxins and a systematic search on the protein data bank revealed the occurrence of a KX-hydrophobic-hydrophobic-P motif that seems to be important for helix stabilization.

Circular dichroism and proteolysis of human β-endorphin in surfactant and lipid solutions

The influence of micelles of sodium dodecyl sulfate, cetyltrimethylammonium bromide, lysophosphatidylcholine and dodecylphosphorylcholine on the content and stability of the ordered structure of human beta-endorphin and its 12-26 fragment has been investigated. The structure was determined by far-ultraviolet circular dichroism and the stability by the resistance of the polypeptide to proteolysis with trypsin and chymotrypsin, monitored by HPLC. The alpha-helix inducing effects of the amphipathic compounds were in the order anionic greater than zwitterionic greater than cationic. The protection against proteolysis was very marked, especially for trypsin, and it was proportional to the alpha-helix inducing potential of amphipathic compounds. However, the lower resistance to proteolysis of the highly structured 12-26 fragment suggests that factors other than secondary structure may be responsible for the resistance to proteolysis.

The design of a specific ligand of HIV gp120.

The crystal structure of CD4 suggested that the C/G38 and C/L44 replacements with the consequent cystine bridge formation are compatible with the native structure of that molecular moiety. As the NQGSF sequence, corresponding to the 39–43 fragment of human CD4 protein, was found to be involved in the HIV gp120 interaction, it has been synthesized in a cyclic form by adding two cysteine residues at the amino and carboxy termini. 1H‐NMR studies show that the predominant solution conformation of cyclo‐[CNQGSFC] is a type II β‐turn centred on the NQGS segment. Structural and dynamic properties of the peptide are also analysed in relation to the in vitro activity.

The Use of Paramagnetic Probes for NMR Investigations of Biomolecular Structures and Interactions

The structure/function relationship in protein is a fundamental topic of investigation in every branch of biotechnology. The possibility of de novo design of transelectronase enzymes to be used in bioelectronic devices implies a good knowledge of folding mechanism and solution structures. Furthermore, the enzyme surface accessibity to substrates is of particular interest. Nuclear magnetic resonance (NMR) is nowadays a well established technique for the investigation of the solution conformation of complex molecules (Wuthrich; Dweck et al., Campbell et al.) and of their mutual interaction processes.