In order to accomplish the selective synthesis of [60]fullerene bisadducts, the reactions of [60]fullerene with compounds in which two α,α‘-dibromo-o-xylene moieties were connected by an oligomethylene chain (n = 2−5) were investigated. By this method, only two isomers (cis-2- and cis-3-isomers) were selectively obtained when n = 2 and 3, while another isomer (e-isomer) was obtained when n = 5. When n = 4, a complex mixture of bisadducts was formed and has not been separated so far. cis-2-Bisadducts have been, for the first time, selectively obtained in the fullerene chemistry. The structures of bisadducts were determined on the basis of 1H, 13C NMR, IR, UV−vis, and mass spectroscopies. According to the NMR experiments, the symmetries of cis-2-, cis-3-, and e-isomers were concluded to be C s , C 2, and C 1, respectively. Chiral cis-3- and e-bisadducts were successfully resolved into the respective enantiomers on a chiral HPLC column, although cis-2-bisadducts only gave a single peak. The UV−vis spectra of cis-2-, cis-3-, and e-bisadducts were remarkably different from one another. Specifically, the e-bisadducts showed a characteristic absorption peak around 420 nm. The cleavage of the oligomethylene chain produced the corresponding [60]fullerene derivatives possessing two phenol moieties. These compounds are applicable to further functionalization.

SummaryCells adapt to oxidative stress by transcriptional activation of genes encoding antioxidants and proteins of other protective roles. A bZIP transcription factor, Pap1, plays a critical role in this process and overexpression of Pap1 confers resistance to various oxidants and drugs in fission yeast. Pap1 temporarily enters the nucleus upon oxidative stress but returns to the cytoplasm once cells adapt to the stress, suggesting that cellular localization regulates Pap1 function. We report here an additional regulatory mechanism that Ubr1 ubiquitin ligase-dependent degradation lowered the Pap1 protein levels. ubr1 cells were causally resistant to hydrogen peroxide because of the increment of Pap1 levels. Pap1 was preferentially degraded in the nucleus where Ubr1 was consistently enriched. Proteolysis was critical to downregulate Pap1 especially when its activation persisted, as constitutively nuclear Pap1 severely inhibited growth in ubr1 mutants. Inactive mutations in the bZIP DNA binding domain stabilized Pap1 but rescued the lethality caused by constitutively active Pap1 in ubr1 mutants. These findings indicate that either nuclear export or Ubr1-mediated proteolysis must be operative to prevent uncontrolled Pap1 function. Coincidental dysfunction in both inhibitory pathways causes lethality because of prolonged activation of Pap1. Ubr1 is a critical regulator for the homeostasis of oxidative stress response.

We report here a new strategy for derivatizing peptides and proteins at the N‐terminus. To achieve this, a nonnatural amino acid was charged onto a tRNA and then enzymatically transferred to a lysine (Lys) unit at the N‐terminus of a peptide or a protein by using L/F‐tRNA‐protein transferase. By using the chemoenzymatic technique, β‐(2‐quinolyl)‐L‐alanine, p‐azido‐L‐phenylalanine, and p‐acetyl‐L‐phenylalanine were introduced to the N‐terminus. The latter two nonnatural amino acids possess bioorthogonal functional groups to which artificial tags can be introduced. Actually, a biotin tag was coupled to the bioorthogonal ketone group of acetylphenylalanine at the N‐terminus of a peptide. N‐terminal‐specific biotinylation and fluorescence derivatization of the bioorthogonal azido‐containing protein or peptide was also carried out based on a [3 + 2] cycloaddition. The enzymatic transfer of a nonnatural amino acid to the N‐terminus of target peptides or proteins was also successfully achieved in the presence of other peptides or crude protein mixtures. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 263–271, 2007. This article was originally published online as an accepted preprint. The ‘Published Online’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Four-base codon strategy was applied to incorporate a fluorophore-quencher pair into specific positions on a single protein; beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap) was employed as a fluorophore and p-nitrophenylalanine (ntrPhe) as a quencher. Their positions were directed by the CGGG/CCCG and GGGC/CCCG four-base codon/anticodon pairs and two doubly mutated streptavidins, i.e., ((52)atnDap, (84)ntrPhe) and ((54)ntrPhe, (84)atnDap) mutants were synthesized through Escherichia coli in vitro protein synthesizing systems. Intramolecular photoinduced electron transfer (ET) was observed as the decrease of intensity in steady-state fluorescence spectroscopy and as the shortening of fluorescence decaytimes. The quenching data indicated that the ET rate reflects the detailed structure of the protein.

The site-specific introduction of a haloacetamide derivative into a designated cysteine on a displaying peptide on a capsid protein (gp10) of bacteriophage T7 has been achieved. This easiest gp10-based thioetherification (10BASEd-T) is carried out in one-pot without side reactions or loss of phage infectivity.

Phage display technology is undoubtedly a powerful tool for affinity selection of target-specific peptide. Commercially available premade phage libraries allow us to take screening in the easiest way. On the other hand, construction of a custom phage library seems to be inaccessible, because several practical tips are absent in instructions. This paper focuses on what should be born in mind for beginners using commercially available cloning kits (Ph.D. with type 3 vector and T7Select systems for M13 and T7 phage, respectively). In the M13 system, Pro or a basic amino acid (especially, Arg) should be avoided at the N-terminus of peptide fused to gp3. In both systems, peptides containing odd number(s) of Cys should be designed with caution. Also, DNA sequencing of a constructed library before biopanning is highly recommended for finding unexpected bias.

No abstract

No abstract