A photochromic azobenzene linker was incorporated as a turn element into an amino acid sequence known to fold into a beta-hairpin structure in aqueous solution. Oligomer formation when the linker was in its thermodynamically favored trans form prohibited structure determination. Light-induced conformational change of the linker to the cis form led to the formation of monomers which exhibited a well-defined beta-hairpin structure as determined by (1)H NMR. The rate of the light-induced cis-to-trans isomerization of the azobenzene-containing peptide was 30% slower compared to the unsubstituted chromophore. These results suggest that suitably substituted azobenzenes can be used as photoinducible turn elements to investigate and control the folding and stability of beta-sheets.

By means of a systematic approach, several green solvent candidates were tested for their feasibility to replace the reprotoxic dimethylformamide (DMF) as a solvent used in solid-phase peptide synthesis (SPPS). According to the results presented in this paper, it is clear that N-butylpyrrolidinone (NBP) is the best green solvent candidate to replace DMF in SPPS.

Photolabile caging groups, including the 1-(2-nitrophenyl)ethyl (NPE) group, have been applied to probe many biological processes, including protein phosphorylation. Although studies with NPE-caged phosphoamino acids have provided valuable information, these investigations have been limited to the use of only one caged species in a single experiment. To expand the scope of these tools, we developed an approach to sequentially uncage two different phosphopeptides in one system, enabling interrogation of multiple phosphorylation events. We present the synthesis of [7-(diethylamino)coumarin-4-yl]methyl (DEACM)-caged phosphorylated serine, threonine, and tyrosine building blocks for Fmoc-based solid phase peptide synthesis to allow convenient incorporation of these residues into peptides and proteins. Exposure of DEACM- and NPE-caged phosphopeptides to 420 nm light selectively releases the DEACM group without affecting the NPE-caged peptide. This then enables a subsequent irradiation event at 365 nm to remove the NPE group and liberate a second phosphopeptide. We demonstrate the versatility of this general sequential uncaging approach by applying it to control the Wip1 phosphatase with two wavelengths of light.

The study presented here consists of three parts. In the first, the ability of a set of differently substituted diazobenzene-based linkers to act as photoswitchable beta-turn building blocks was assessed. A 12-residue peptide known to form beta-hairpins was taken as the basis for the modeling process. The central (beta-turn) residue pair was successively replaced by six symmetrically ((o,o), (m,m), or (p,p)) substituted (aminomethyl/carboxymethyl or aminoethyl/carboxyethyl) diazobenzene derivatives leading to a set of peptides with a photoswitchable backbone conformation. The folding behavior of each peptide was then investigated by performing molecular dynamics simulations in water (4 ns) and in methanol (10 ns) at room temperature. The simulations suggest that (o,o)- and (m,m)-substituted linkers with a single methylene spacer are significantly better suited to act as photoswitchable beta-turn building blocks than the other linkers examined in this study. The peptide containing the (m,m)-substituted linker was synthesized and characterized by NMR in its cis configuration. In the second part of this study, the structure of this peptide was refined using explicit-solvent simulations and NOE distance restraints, employing a variety of refinement protocols (instantaneous and time-averaged restraining as well as unrestrained simulations). We show that for this type of systems, even short simulations provide a significant improvement in our understanding of their structure if physically meaningful force fields are employed. In the third part, unrestrained explicit-solvent simulations starting from either the NMR model structure (75 ns) or a fully extended structure (25 ns) are shown to converge to a stable beta-hairpin. The resulting ensemble is in good agreement with experimental data, indicating successful structure prediction of the investigated hairpin by classical explicit-solvent molecular dynamics simulations.

Since the early work of Anfinsen, structural biology has been influenced by the dogma that proteins fold into unique structures dictated by their amino acid sequence.[1] However, the recent observation that prion proteins undergo spontaneous conversion from well-folded and soluble helical entities into insoluble b-sheet-containing aggregates suggests that this view is overly simplistic.[2] It also raises the intriguing possibility that conformational switches might be designed to transform proteins deliberately from one unique structure into another unique structure. Such switches could be quite valuable, for example, for studying folding in the absence of denaturants or for controlling receptor function or enzyme activity.Photochromic compounds, which undergo large conformational changes when exposed to light of an appropriate wavelength, are interesting in this context as they might permit reversible conformational control. Of the many photochromic systems that have been described, azobenzenes are well suited for this type of application as they have been extensively characterized and are readily synthesized. Irradiation at the wavelength of the p!p * transition converts the thermodynamically favored trans to the cis isomer, whereas the reverse process can be achieved either thermally or by irradiation at the wavelength of the n!p * transition. Azobenzenes have been introduced into many peptides and proteins, both into side chains [3,4] and into the main chain. [5,6] For example, our research group [7] and Moroder and coworkers [8] have shown that the meta-substituted Fmoc-protected linker 1 (Scheme 1) can be inserted into a polypeptide backbone through solid-phase peptide synthesis and, in its cis configuration, serve as the b-turn-inducing segment in bhairpin peptides. Thermally induced isomerization from cis-1 to trans-1 results in unfolding of the hairpin.In this study, we have extended our work to larger helical polypeptides. As a model system, we chose a derivative of avian pancreatic polypeptide (aPP). The 36 residue hormone was one of the first peptides for which a crystal structure was available.[9] It consists of a C-terminal a helix (residues 14-31) connected through a b turn (residues 9-13) to an N-terminal type-II polyproline helix, a structural motif known as the PP fold. In the solid state and in solution, two aPP polypeptides further associate to form a symmetric dimer.[10] Despite its small size, aPP thus displays the features of much larger proteins, that is, it a possesses secondary as well as tertiary and quaternary structure.To allow switching of the aPP peptide from a back-folded (PP-folded) to an extended form, we replaced the b-turn segment between the helices, comprising residues Asp 10-Asp 11-Ala 12, with 1. The resulting molecule, which exhibits similar helicity to natural aPP (as determined by circular dichroism spectroscopy), is readily interconverted between the cis and trans forms upon irradiation. At thermodynamic equilibrium, the trans/cis ratio is 90:10, but the fraction of cis...