Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington's disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely β-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for β-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended β-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing β-strands to more than one sheet and an antiparallel assembly of strands within β-sheets.

Serine/arginine-rich (SR) proteins are important players in RNA metabolism and are extensively phosphorylated at serine residues in RS repeats. Here, we show that phosphorylation switches the RS domain of the serine/arginine-rich splicing factor 1 from a fully disordered state to a partially rigidified arch-like structure. Nuclear magnetic resonance spectroscopy in combination with molecular dynamics simulations revealed that the conformational switch is restricted to RS repeats, critically depends on the phosphate charge state and strongly decreases the conformational entropy of RS domains. The dynamic switch also occurs in the 100 kDa SR-related protein hPrp28, for which phosphorylation at the RS repeat is required for spliceosome assembly. Thus, a phosphorylation-induced dynamic switch is common to the class of serine/arginine-rich proteins and provides a molecular basis for the functional redundancy of serine/arginine-rich proteins and the profound influence of RS domain phosphorylation on protein-protein and protein-RNA interactions.

A representative series of diphosphine monophosphonium salts [1-Ph 2 P(C 10 H 6 )-8-PRPh 2 ] + X ± (2 b: R = H, X = CF 3 SO 3 ; 4: R = Me, X = CF 3 SO 3 ; 5: R = C 6 H 5 CH 2 = Bn, X = Br) has been prepared by treatment of 1,8-bis(diphenylphosphino)naphthalene (dppn, 1) with stoichiometric amounts of HSO 3 CF 3 or CH 3 SO 3 CF 3 in CH 2 Cl 2 at +20°C and with C 6 H 5 CH 2 Br in toluene at +80°C. Their X-ray crystal structures show that there is no evidence for dative P ® P + interactions. Instead, steric repulsion deflects the substituent groups to opposite faces of the naphthalene plane [splay angles: +11.4°(2 b), +13.6°(4); +16.7°(5)]. In solution 2 b, 4, and 5 were dynamic according to 31 P, 13 C, and 1 H NMR spectroscopy. The fluxionality of 2 b involves rapid intramolecular proton exchange between the two phosphorus atoms, which slows down at low temperature, whereas the dynamic behaviour of 4 and 5 is interpreted in terms of hindered rotation of the bulky RPh 2 P + groups (R = Me or Bn) about the P±C(naphthyl) bond. Treatment of 1,8-bis(diphenylphosphoryl)naphthalene (dppnO 2 , 6) with HSO 3 CF 3 gave the protonated bis(phosphine oxide), as the triflate salt, dppnO 2 H + CF 3 SO 3 ± (7). The X-ray structure analysis of 7 revealed a highly strained molecule (P1´´´P2 365.5 pm) in which the P=O bonds point to the same face of the naphthalene plane to accommodate the proton. All isolated compounds were characterised by a combination of 31 P, 1 H, and 13 C NMR spectroscopy, IR spectroscopy (7), mass spectrometry and elemental analysis.Phosphonium-Salze von 1,8-Bis(diphenylphosphino)naphthalin: Moleku È lstrukturen und NMR-spektroskopische Untersuchungen Inhaltsu È bersicht. Durch Umsetzung von 1,8-Bis(diphenylphosphino)naphthalin (dppn, 1) mit sto È chiometrischen Mengen HSO 3 CF 3 oder CH 3 SO 3 CF 3 in CH 2 Cl 2 bei +20°C und mit C 6 H 5 CH 2 Br in Toluol bei +80°C wurde eine repra È-sentative Reihe von Diphosphin-Monophosphoniumsalzen [1-Ph 2 P(C 10 H 6 )-8-PRPh 2 ] + X ± (2 b: R = H, X = CF 3 SO 3 ; 4: R = Me, X = CF 3 SO 3 ; 5: R = C 6 H 5 CH 2 = Bn, X = Br) hergestellt. Die Ro È ntgenstrukturanalysen ergeben keine Hinweise auf dative, bindende P ® P + Wechselwirkungen. Stattdessen fu È hrt sterische Abstoûung zu einer Ablenkung der Substituenten auf entgegengesetzte Seiten der NaphthalinEbene [Spreizwinkel: +11.4°(2 b), +13.6°(4); +16.7°(5)]. In Lo È sung zeigten 2 b, 4 und 5 bei 31 P-, 13 C-und 1 H-NMRspektroskopischen Untersuchungen dynamisches Verhalten. Bei 2 b handelt es sich um einen schnellen, intramolekularen Protonenaustausch zwischen den beiden Phosphoratomen, der bei niedrigen Temperaturen verlangsamt ist, wa È hrend das dynamische Verhalten von 4 und 5 durch gehinderte Rotation der sperrigen RPh 2 P + Gruppen (R = Me oder Bn) um die P±C(Naphthyl) Bindung erkla È rt werden kann. Die Umsetzung von 1,8-Bis(diphenylphosphoryl)naphthalin (dppnO 2 , 6) mit HSO 3 CF 3 fu È hrte zum protonierten Bis(phosphinoxid), in Form des Triflatsalzes, dppnO 2 H + CF 3 SO 3 ± (7). Die Ro È ntgenstrukturanalyse von...

During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.HIV-1 entry (reviewed in reference 2) is a multistep process initiated by binding of the outer envelope (Env) glycoprotein gp120 to the CD4 receptor. This results in conformational changes leading to exposure of CD4-induced (CD4i) epitopes for the subsequent obligatory interaction with the chemokine receptor CCR5 or CXCR4. Further conformational changes in gp120 activate the fusion peptide at the N terminus of the transmembrane glycoprotein gp41 and lead to the assembly of a six-helix bundle structure that triggers membrane fusion and internalization of the viral particles. This sequential HIV-1 entry process allows the virus to protect the functionally important and highly conserved Env entry epitopes by limiting their exposure to antibodies which may neutralize the infection (35). The complex HIV-1 entry process offers the opportunity for therapeutic intervention at multiple steps. Thus, the first HIV-1 entry inhibitor approved by the FDA (T20, enfuvirtide [Fuzeon]) is a peptide inhibiting the very last step of entry, i.e., membrane fusion, by interfering with the six-helix bundle formation (39). A second entry inhibitor (maraviroc [Selzentry]), targeting CCR5, was approved by the FDA in 2007 (46). This drug, like others whose development had to be discontinued due to liver toxicities, is a CCR5 antagonist, and its use is restricted to drug-experienced HIV-1...

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