Combining modern synthetic and molecular biology toolkits, native chemical ligation and expressed protein ligation enables robust access to modified proteins.

Amide proton NMR signals from the N-terminal domain of monomeric a-synuclein (aS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in a-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of aS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N-and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Ca chemical shift data point to <10% residual a-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in 1 H-15 N HSQC spectra of aS encapsulated within living Escherichia coli cells. Our finding that temperature effects on aS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that aS remains predominantly unfolded at physiological temperature and pH-an important conclusion for mechanistic models of the association of aS with membranes and fibrils. Abbreviations: aS, a-synuclein; ct, constant time; CLEANEX, clean chemical exchange; DSS, 2,2-Dimethyl-2-silapentane-5-sulfonic acid; HSQC, heteronuclear single quantum coherence spectroscopy; GM1, monosialotetrahexosylganglioside; R2, transverse relaxation rate (1/ T2); R2 ex , chemical exchange contribution to R2; SDS, sodium dodecyl sulfate.Article and publication are at http://www.proteinscience.org/cgi

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the Anopheles mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated in vivo. To test this prediction, insect cell expression of two anophelin proteins, from Anopheles albimanus and Anopheles gambiae, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation–desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from A. albimanus exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.

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Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.