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.

The oncoprotein MDM2 inhibits the tumor suppressor protein p53 by binding to the p53 transactivation domain. The p53 gene is inactivated in many human tumors either by mutations or by binding to oncogenic proteins. In some tumors, such as soft tissue sarcomas, overexpression of MDM2 inactivates an otherwise intact p53, disabling the genome integrity checkpoint and allowing cell cycle progression of defective cells. Disruption of the MDM2/p53 interaction leads to increased p53 levels and restored p53 transcriptional activity, indicating restoration of the genome integrity check and therapeutic potential for MDM2/p53 binding antagonists. Here, we show by multidimensional NMR spectroscopy that chalcones (1,3-diphenyl-2-propen-1-ones) are MDM2 inhibitors that bind to a subsite of the p53 binding cleft of human MDM2. Biochemical experiments showed that these compounds can disrupt the MDM2/p53 protein complex, releasing p53 from both the p53/MDM2 and DNA-bound p53/MDM2 complexes. These results thus offer a starting basis for structure-based drug design of cancer therapeutics.

Femtosecond time-resolved spectroscopy on model peptides with built-in light switches combined with computer simulation of light-triggered motions offers an attractive integrated approach toward the understanding of peptide conformational dynamics. It was applied to monitor the light-induced relaxation dynamics occurring on subnanosecond time scales in a peptide that was backbone-cyclized with an azobenzene derivative as optical switch and spectroscopic probe. The femtosecond spectra permit the clear distinguishing and characterization of the subpicosecond photoisomerization of the chromophore, the subsequent dissipation of vibrational energy, and the subnanosecond conformational relaxation of the peptide. The photochemical cis͞trans-isomerization of the chromophore and the resulting peptide relaxations have been simulated with molecular dynamics calculations. The calculated reaction kinetics, as monitored by the energy content of the peptide, were found to match the spectroscopic data. Thus we verify that all-atom molecular dynamics simulations can quantitatively describe the subnanosecond conformational dynamics of peptides, strengthening confidence in corresponding predictions for longer time scales.