Christian Renner

Azobenzene as conformational switch in model peptides

The photoinduced isomerization of azobenzene between the extended (trans) and compact (cis) conformations is reversibly triggered by light of two differing wavelengths. The resulting changes in molecular geometry have been extensively utilized to photoswitch transformations in chemical species reversibly for applications in optoelectronic devises as well as to photocontrol conformational states in (bio)polymers. The high isomerization yield, remarkable photostability and ultrafast kinetics (few ps) of azobenzene are well suited for the design of small, defined model systems that allow detailed folding studies to be carried out both experimentally and theoretically on the same molecules. In our and other laboratories such systems were recently obtained with cyclic peptides of defined conformational preferences as well as with α‐helical and β‐hairpin peptides. These should, by comparison of simulation and experiment, permit an assessment and improvement of the theoretical description on the one hand and a detailed interpretation of the ultrafast conformational dynamics on the other. The phototriggered changes in conformational states lead to concurrent changes in biophysical properties that can be exploited in the photocontrol of biochemical and biological events, as exemplarily discussed with redox‐active cyclic bis‐cysteinyl peptides and receptor ligands.

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions.

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 β‐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.

Ultrafast spectroscopy reveals subnanosecond peptide conformational dynamics and validates molecular dynamics simulation.

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 cis/trans-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.

Picosecond conformational transition and equilibration of a cyclic peptide

Ultrafast IR spectroscopy is used to monitor the nonequilibrium backbone dynamics of a cyclic peptide in the amide I vibrational range with picosecond time resolution. A conformational change is induced by means of a photoswitch integrated into the peptide backbone. Although the main conformational change of the backbone is completed after only 20 ps, the subsequent equilibration in the new region of conformational space continues for times >16 ns. Relaxation and equilibration processes of the peptide backbone occur on a discrete hierarchy of time scales. Albeit possessing only a few conformational degrees of freedom compared with a protein, the peptide behaves highly nontrivially and provides insights into the complexity of fast protein folding.

Photomodulation of the Conformation of Cyclic Peptides with Azobenzene Moieties in the Peptide Backbone

The cisright harpoon over left harpoon trans photoisomerization of the azobenzene building block 4-(4-aminophenylazo)benzoic acid incorporated in a cyclic peptide (see scheme) facilitated a two-state transition of the peptide chain from a rigid constrained conformation in the trans isomer into the largely free conformational space of the cis isomer.

The Extracellular Human Melanoma Inhibitory Activity (Mia) Protein Adopts an SH3 Domain-Like Fold.

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain‐like fold in solution, with two perpendicular, antiparallel, three‐ and five‐stranded β‐sheets. In contrast to known structures with the SH3 domain fold, MIA is a single‐domain protein, and contains an additional antiparallel β‐sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain‐like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin α4β1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.

Practical aspects of the 2D 15N-{1H}-NOE experiment

The heteronuclear 15N-NOE experiment was extensively tested with respect to statistical and systematic experimental error. The dependence of signal intensity in the NOE experiment and in the reference experiment on the saturation and relaxation time was experimentally investigated. The statistics of the experimental values were accessed by numerous repetitions of identical set-ups. As a model system a protein of typical size for NMR studies was chosen, i.e., a 120 amino acid residues containing fragment of the F-actin binding gelation factor (ABP-120). The fragment exhibits fast dynamics that are accessible with the 15N-NOE experiment with various amplitudes. The results of this study show that commonly used parameters are only adequate for accurate measurement of motions with moderate amplitude. Highly flexible parts require longer delay times and thus more experimental time than commonly used. On the other hand, a qualitative or semi-quantitative assessment of a proteins mobility on fast times scales can be obtained from rapidly recorded experiments with unusual short delay times. The findings of this study are of equal importance for highly accurate measurement of the 15N-NOE as well as for quick identification of mobile or even unstructured residues/parts of a protein.

Light-triggered β-hairpin folding and unfolding

A light-switchable peptide is transformed with ultrashort pulses from a β-hairpin to an unfolded hydrophobic cluster and vice versa. The structural changes are monitored by mid-IR probing. Instantaneous normal mode analysis with a Hamiltonian combining density functional theory with molecular mechanics is used to interpret the absorption transients. Illumination of the β-hairpin state triggers an unfolding reaction that visits several intermediates and reaches the unfolded state within a few nanoseconds. In this unfolding reaction to the equilibrium hydrophobic cluster conformation, the system does not meet significant barriers on the free-energy surface. The reverse folding process takes much longer because it occurs on the time scale of 30 μs. The folded state has a defined structure, and its formation requires an extended search for the correct hydrogen-bond pattern of the β-strand.

The Core Structure of TMC-95A Is a Promising Lead for Reversible Proteasome Inhibition

Correspondingly,great attention has recently been paid to the discovery ofpotentandselectiveproteasomeinhibitorsbystructure-baseddesignornaturalproductscreeningapproaches.Mostofthesynthetic inhibitors consisting of peptide aldehydes, boro-nates, and vinylsulfones, as well as the natural productslactacystinandepoxymicinsinhibitinamoreorlessselectivemanner the proteasome by reaction with the N-terminalthreonineresidue(forarecentreviewseeref.[5]).Anotableexceptionisthehighlyselectiveandcompetitiveproteasomeinhibitor TMC-95A, which was isolated from the fermenta-tionbrothofApiosporamontagneiSacc.TC1093.

Ultrafast Conformational Dynamics in Cyclic Azobenzene Peptides of Increased Flexibility

Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the cis to trans and the trans to cis photoreactions. For the cis to trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the trans to cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps-1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10 ns.