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The LAH4 family of histidine-rich peptides exhibits potent antimicrobial and DNA transfection activities, both of which require interactions with cellular membranes. The bilayer association of the peptides has been shown to be strongly pH-dependent, with in-planar alignments under acidic conditions and transmembrane orientations when the histidines are discharged. Therefore, we investigated the pH- and temperature-dependent conformations of LAH4 in DPC micellar solutions and in a TFE/PBS solvent mixture. In the presence of detergent and at pH 4.1, LAH4 adopts helical conformations between residues 9 and 24 concomitantly with a high hydrophobic moment. At pH 6.1, a helix-loop-helix structure forms with a hinge encompassing residues His¹⁰-Ala¹³. The data suggest that the high density of histidine residues and the resulting electrostatic repulsion lead to both a decrease in the pK values of the histidines and a less stable α-helical conformation of this region. The hinged structure at pH 6.1 facilitates membrane anchoring and insertion. At pH 7.8, the histidines are uncharged and an extended helical conformation including residues 4-21 is again obtained. LAH4 thus exhibits a high degree of conformational plasticity. The structures provide a stroboscopic view of the conformational changes that occur during membrane insertion, and are discussed in the context of antimicrobial activity and DNA transfection., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Whereas bacterial expression systems are widely used for production of uniformly or selectively (15)N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively (15)N-labeled proteins in insect cells. The quantities of (15)N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the (15)N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.

Human macrophage migration inhibitory factor is a 114 amino acid protein that belongs to the family of immunologic cytokines. Assignments of 1H, 15N, and 13C resonances have enabled the determination of the secondary structure of the protein, which consists of two alpha-helices (residues 18-31 and 89-72) and a central four-stranded beta-sheet. In the beta-sheet, two parallel beta-sheets are connected in an antiparallel sense. From the total of three cysteines present in the primary structure of MIF, none was found to form disulfide bridges. 1H-15N heteronuclear T1, T2, and steady-state NOE measurements indicate that the backbone of MIF exists in a rigid structure of limited conformational flexibility (on the nanosecond to picosecond time scale). Several residues located in the loop regions and at the N termini of two helices exhibit internal motions on the 1-3 ns time scale. The capacity to bind glutathione was investigated by titration of a uniform 15N-labeled sample and led us to conclude that MIF has, at best, very low affinity for glutathione.