Synthesis and Photoisomerization of Substituted Dibenzofulvene Molecular Rotors.

The synthesis, spectral and structural characterization, and photoisomerization of a family of 2-substituted dibenzofulvene molecular actuators based on (2,2,2-triphenylethylidene)fluorene (TEF) are reported. The 2-substituted species investigated are nitro (NTEF), cyano (CTEF), and iodo (ITEF). X-ray structures of these three compounds and three intermediates were determined to assign alkene configuration and investigate the effects of the 2-substituents on steric gearing. The addition-elimination reaction of Z-9 with trityl anion to form Z-10 proceeded with complete retention of configuration. Rates of photoisomerization were measured at irradiation wavelengths between 266-355 nm in acetonitrile/dioxane solutions at room temperature. Photoisomerization quantum yields (φ) were calculated by means of a mathematical model that accounts for a certain degree of photodecomposition in the cases of CTEF and ITEF. Quantum yields vary significantly with substituent, having maximum values of φ=0.26 for NTEF, 0.39 for CTEF, and 0.50 for ITEF. NTEF is photochemically robust and has a large quantum yield for photoisomerization in the near-UV, making it a particularly promising drive rotor moiety for light-powered molecular devices.

Differential binding between PatS C-terminal peptide fragments and HetR from Anabaena sp. PCC 7120.

Heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120 occurs at regular intervals under nitrogen starvation and is regulated by a host of signaling molecules responsive to availability of fixed nitrogen. The heterocyst differentiation inhibitor PatS contains the active pentapeptide RGSGR (PatS-5) at its C-terminus considered the minimum PatS fragment required for normal heterocyst pattern formation. PatS-5 is known to bind HetR, the master regulator of heterocyst differentiation, with a moderate affinity and a submicromolar dissociation constant. Here we characterized the affinity of HetR for several PatS C-terminal fragments by measuring the relative ability of each fragment to knockdown HetR binding to DNA in electrophoretic mobility shift assays and using isothermal titration calorimetry (ITC). HetR bound to PatS-6 (ERGSGR) >30 times tighter (K(d) = 7 nM) than to PatS-5 (K(d) = 227 nM) and >1200 times tighter than to PatS-7 (DERGSGR) (K(d) = 9280 nM). No binding was detected between HetR and PatS-8 (CDERGSGR). Quantitative binding constants obtained from ITC measurements were consistent with qualitative results from the gel shift knockdown assays. CW EPR spectroscopy confirmed that PatS-6 bound to a MTSL spin-labeled HetR L252C mutant at a 10-fold lower concentration compared to PatS-5. Substituting the PatS-6 N-terminal glutamate to aspartate, lysine, or glycine did not alter binding affinity, indicating that neither the charge nor size of the N-terminal residue's side chain played a role in enhanced HetR binding to PatS-6, but rather increased binding affinity resulted from new interactions with the PatS-6 N-terminal residue peptide backbone.

Solution structure of the HIV-1 exon splicing silencer 3.

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impair HIV-1 replication. Genome splicing in HIV-1 is tightly regulated by the dynamic assembly/disassembly of trans host factors with cis RNA control elements. The host protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, regulates splicing at several highly conserved HIV-1 3' splice sites by binding 5'-UAG-3' elements embedded within regions containing RNA structure. The physical determinants of hnRNP A1 splice site recognition remain poorly defined in HIV-1, thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the three-dimensional structure of the exon splicing silencer 3 (ESS3) from HIV-1 has been determined using NMR spectroscopy. ESS3 adopts a 27-nucleotide hairpin with a 10-bp A-form stem that contains a pH-sensitive A(+)C wobble pair. The seven-nucleotide hairpin loop contains the high-affinity hnRNP-A1-responsive 5'-UAGU-3' element and a proximal 5'-GAU-3' motif. The NMR structure shows that the heptaloop adopts a well-organized conformation stabilized primarily by base stacking interactions reminiscent of a U-turn. The apex of the loop is quasi-symmetric with UA dinucleotide steps from the 5'-GAU-3' and 5'-UAGU-3' motifs stacking on opposite sides of the hairpin. As a step towards understanding the binding mechanism, we performed calorimetric and NMR titrations of several hnRNP A1 subdomains into ESS3. The data show that the UP1 domain forms a high-affinity (K(d)=37.8±1.1 nM) complex with ESS3 via site-specific interactions with the loop.

Evidence for direct binding between HetR from Anabaena sp. PCC 7120 and PatS-5.

HetR, master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120, stimulates heterocyst differentiation via transcriptional autoregulation and is negatively regulated by PatS and HetN, both of which contain the active pentapeptide RGSGR. However, the direct targets of PatS and HetN remain uncertain. Here, we report experimental evidence for direct binding between HetR and the C-terminal RGSGR pentapeptide, PatS-5. Strains with a hetR allele coding for conservative substitutions at residues 250-256 had altered patterns of heterocysts and, in some cases, reduced sensitivity to PatS-5. Cysteine scanning mutagenesis coupled with electron paramagnetic resonance (EPR) spectroscopy showed quenching of spin label motion at HetR amino acid 252 upon titration with PatS-5, indicating direct binding of PatS-5 to HetR. Gel shift assays indicated that PatS-5 disrupted binding of HetR to a 29 base pair inverted-repeat-containing DNA sequence upstream from hetP. Double electron-electron resonance EPR experiments confirmed that HetR existed as a dimer in solution and indicated that PatS-5 bound to HetR without disrupting the dimer form of HetR. Isothermal titration calorimetry experiments corroborated direct binding of PatS-5 to HetR with a K(d) of 227 nM and a 1:1 stoichiometry. Taken together, these results indicated that PatS-5 disrupted HetR binding to DNA through a direct HetR/PatS interaction. PatS-5 appeared to either bind in the vicinity of HetR amino acid L252 or, alternately, to bind in a remote site that leads to constrained motion of this amino acid via an allosteric effect or change in tertiary structure.