Shou-Liang Dong

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.

Toward Semisynthetic Lipoproteins by Convergent Strategies Based on Click and Ligation Chemistry

Cell-membrane proteins are anchored to the lipid bilayer by single or multiple insertion of transmembrane helices or by regioselective single or dual lipidation in coand post-translational enzymatic processes, including acylation with fatty acids, prenylation, and rather commonly C-terminal amidation with glycosylphosphatidylinositols (GPI). Procedures for native and neolipidation of peptides have been comprehensively reviewed, and, more recently, even the synthesis of complex GPIs has been reported. However, lipidation of proteins at defined sites and particularly grafting of GPIs or related mimetic structures to the C termini of proteins still represent formidable long-term goals of chemistry and molecular biology. So far, lipoproteins have been obtained by chemical ligation of synthetic lipopeptides with recombinant protein fragments by the highly selective and efficient maleinimide/thiol addition reaction, as shown for the RAS protein, for example, or by total synthesis through orthogonal protection schemes for regioselective lipidation of side-chain amino groups, for example, with 1,2-dipalmitoyl-glycero-3-succinate. In view of the recently developed efficient procedures for the semisynthesis of proteins by native-chemical [6] and expressed-protein ligation, we have performed model studies toward C-terminal lipidation of proteins by exploiting the copper(i)-catalyzed Huisgen’s 1,3-dipolar cycloaddition of terminal alkynes to azides to form a stable triazole product and the transthioesterification between peptide thioesters and N-cysteinyl-lipopeptides followed by intramolecular S!N acyl shift as the synthetic strategy set forth in Scheme 1. Aside from validating the methodology of a combined click and ligation chemistry, incubation of HeLa cells with the micellar solution of the lipopeptide confirmed its fast uptake, as visualized by confocal fluorescence microscopy. It is well established that dual vicinal lipid chains, as present in the di-fattyacyl glycerol moiety of natural GPI anchors are required for an almost irreversible capture of peptides and proteins by lipid bilayers. Correspondingly, to properly mimic the GPI anchor, phosphatidylethanolamine was converted into the corresponding azide 1 by CuSO4-catalyzed diazotransfer with triflyl azide to produce the key intermediate for subsequent application of the click chemistry. The crystalline azide 1 was then used for the 1,3-cycloaddition reaction with the Sprotected model peptide 2, which contained a C-terminal propargylglycine (Pra) residue as suitable reaction partner (Scheme 1). The azide–alkyne cycloaddition was performed with CuI as catalyst in organic solvent, and the lipopeptide derivative 3 was isolated by silica gel chromatography in yields of 70–75 %. Upon removal of the acid-labile S-trityl and NBoc groups from 3 with TFA, the subsequent native chemical ligation of the cysteinyl-lipopeptide with N-dansylor N-rhodamine B-labeled Gly-Pro-Gly-Gly-SPh ester 4 was performed in micellar solutions of 2 % octyl-b-d-glucopyranoside. Ligation was found to proceed smoothly in the presence of tris(2-carboxyethyl)phosphine (TCEP) if excess of the thioesters was carefully avoided to prevent bisacylation as a side reaction. HPLC served to isolate the fluorescence-labeled lipopeptides 5 a,b in yields of 60–70 % as analytically well-characterized compounds, as shown in Figure 1 for compound 5 b. The lipopeptide 6 was obtained in practically quantitative yield by treatment of the C-terminal propargylglycine residue with azide 1 in aqueous–organic media and in the presence of [a] H.-J. Musiol, Dr. S. Dong, Dr. M. Kaiser, Prof. Dr. L. Moroder Max-Planck-Institut f r Biochemie Am Klopferspitz 18, 82152 Martinsried (Germany) Fax. : (+ 49) 89-8578-2847 E-mail : moroder@biochem.mpg.de [b] Dipl.-Phys. R. Bausinger, Dr. A. Zumbusch Institut f r physikalische Chemie Butenandtstraße 5–13, 81377 M nchen (Germany) [c] Dr. U. Bertsch Zentrum f r Neuropathologie und Prionforschung, LMU M nchen Feodor Lynen Straße 23, 81377 M nchen (Germany)

Folding and Unfolding of Light-Triggered beta-Hairpin Model Peptides

Ultrafast spectroscopy in the visible and mid-infrared is used to study the reaction dynamics of two light-triggered model peptides containing an azobenzene derivative as a switching element. One model peptide, the AzoTrpZip2, forms a β-hairpin structure in the cis form of the chromophore. This peptide is compared to the core structure consisting of the chromophore and the two flanking amino acid residues, used as a minimal model. This combination of experiments performed in different spectral ranges on peptides of different sizes allows for improved insight into light triggered reaction dynamics. The kinetics observed for the core structure are directly connected to the switching process of the chromophore and are finished on the 10 ps time scale. The trans-to-cis reaction of AzoTrpZip2, leading to the formation of the β-hairpin structure involves ultrafast processes on the 100 ps time scale, which are directly related to the relaxation of the strain between the isomerized molecular switch and the two peptide strands. IR-signatures characteristic for changes in interstrand interactions are absent on the <1 ns time scale. Thus folding into the β-hairpin structure occurs on a much longer time scale. In the cis-to-trans unfolding reaction, all IR signatures related to changes in interstrand interactions occur within 1 ns, in a time range where visible spectroscopy reveals the final decay of the intramolecular strain. Apparently unfolding of AzoTrpZip2 is to a large extent a fast, driven process.

Convenient syntheses of homopropargylglycine

An improved classic Strecker synthesis was elaborated leading to racemic homopropargylglycine (Hpg) in 61% overall yield, while an asymmetric Strecker reaction produced Hpg and the higher homolog 2‐aminohept‐6‐ynoic acid in significantly higher yields and over 80% ee. Copyright

Protein Iodination by Click Chemistry

Two steps to click iodine: We have developed a two‐step reaction for protein iodination using click chemistry. With this method, which is summarized in the scheme, covalent attachment of a stable iodine‐containing aromatic azide moiety to an alkyne‐containing protein was achieved.