Applying γ‐Substituted Prolines in the Foldon Peptide: Polarity Contradicts Preorganization

Rational choice of chemical modifications to proline residues allows the preorganization principle to be exploited for more stable assembly of the foldon domain as a tag for trimerization. With systematic knowledge of how chemical and steric variations of the ring substituents affect the relative stabilities of exo and endo puckers, the preorganization principle should then be usable in biotechnologically synthesized foldon mutants and applicable for protein tagging elsewhere.     

Synthesis and Analysis of Specific Covalent Inhibitors of endo‐Xyloglucanases

A series of N‐bromoacetylglycosylamines and bromoketone C‐glycosides were synthesised from complex xyloglucan oligosaccharide (XyGO) scaffolds as specific active‐site affinity labels for endo‐xyloglucanases. Compounds based on XXXG (Xyl₃Glc₄) and XLLG (Xyl₃Glc₄Gal₂) oligosaccharides exhibited strikingly higher affinities and higher rates of irreversible inhibition than known cellobiosyl and new lactosyl disaccharide congeners when tested with endo‐xyloglucanases from two distinct glycoside hydrolase (GH) families. Intact‐protein mass spectrometry indicated that inactivation with XyGO derivatives generally resulted in a 1:1 labelling stoichiometry. Together, these results indicate that XyGO‐based affinity reagents have significant potential as inhibitors and proteomic reagents for the identification and analysis of diverse xyloglucan‐active enzymes in nature, to facilitate industrial enzyme applications.     

Advanced Mutasynthesis Studies on the Natural α‐Pyrone Antibiotic Myxopyronin from Myxococcus fulvus

Myxopyronin is a natural α‐pyrone antibiotic from the soil bacterium Myxococcus fulvus Mx f50. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by binding to a part of the enzyme not targeted by the clinically used rifamycins. This mode of action makes myxopyronins promising molecules for the development of novel broad‐spectrum antibacterials. We describe the derivatization of myxopyronins by an advanced mutasynthesis approach as a first step towards this goal. Site‐directed mutagenesis of the biosynthetic machinery was used to block myxopyronin biosynthesis at different stages. The resulting mutants were fed with diverse precursors that mimic the biosynthetic intermediates to restore production. Mutasynthon incorporation and production of novel myxopyronin derivatives were analyzed by HPLC‐MS/MS. This work sets the stage for accessing numerous myxopyronin derivatives, thus significantly expanding the chemical space of f α‐pyrone antibiotics.     

Rapid Parallel Synthesis of Bioactive Folded Cyclotides by Using a Tea‐Bag Approach

We report here the first rapid parallel production of bioactive folded cyclotides by using Fmoc‐based solid‐phase peptide synthesis in combination with a “tea‐bag” approach. Using this approach, we efficiently synthesized 15 analogues of the CXCR4 antagonist cyclotide MCo‐CVX‐5c. Cyclotides were synthesized in a single‐pot, cyclization/folding reaction in the presence of reduced glutathione. Natively folded cyclotides were quickly purified from the cyclization/folding crude mixture by activated thiol Sepharose‐based chromatography. The different folded cyclotide analogues were then tested for their ability to inhibit the CXCR4 receptor in a cell‐based assay. The results indicated that this approach can be used for the efficient chemical synthesis of libraries of cyclotides with improved biological properties that can be easily interfaced with solution or cell‐based assays for rapid screening.     

Enzymatic Evidence for a Revised Congocidine Biosynthetic Pathway

Naturally produced pyrrolamides, such as congocidine, are nonribosomal peptides that bind to the minor groove of DNA. Efforts to delineate the biosynthetic machinery responsible for their assembly have mainly employed genetic methods, and the enzymes responsible for their biosynthesis remain largely uncharacterized. We report the biochemical characterization of four proteins involved in congocidine formation: the adenylation‐thiolation (A–T) di‐domain Cgc18(1–610), its MbtH‐like partner SAMR0548, the AMP‐binding enzyme Cgc3*, and the T domain Cgc19. We assayed the ATP‐dependent activation of various commercially available and chemically synthesized compounds with Cgc18(1–610) and Cgc3*. We report the revised substrate specificities of Cgc18(1–610) and Cgc3*, and loading of 4‐acetamidopyrrole‐2‐carboxylic acid onto Cgc19. Based on these biochemical studies, we suggest a revised congocidine biosynthetic pathway.     

Orthogonal Translation Meets Electron Transfer: In Vivo Labeling of Cytochrome c for Probing Local Electric Fields

Cytochrome c (cyt c), a redox protein involved in diverse fundamental biological processes, is among the most traditional model proteins for analyzing biological electron transfer and protein dynamics both in solution and at membranes. Studying the role of electric fields in energy transduction mediated by cyt c relies upon appropriate reporter groups. Up to now these had to be introduced into cyt c by in vitro chemical modification. Here, we have overcome this restriction by incorporating the noncanonical amino acid p‐cyanophenylalanine (pCNF) into cyt c in vivo. UV and CD spectroscopy indicate preservation of the overall protein fold, stability, and heme coordination, whereas a small shift of the redox potential was observed by cyclic voltammetry. The C≡N stretching mode of the incorporated pCNF detected in the IR spectra reveals a surprising difference, which is related to the oxidation state of the heme iron, thus indicating high sensitivity to changes in the electrostatics of cyt c.     

Aggregation‐Prone Amyloid‐β⋅CuII Species Formed on the Millisecond Timescale under Mildly Acidic Conditions

Metal ions and their interaction with the amyloid beta (Aβ) peptide might be key elements in the development of Alzheimer's disease. In this work the effect of Cu^II on the aggregation of Aβ is explored on a timescale from milliseconds to days, both at physiological pH and under mildly acidic conditions, by using stopped‐flow kinetic measurements (fluorescence and light‐scattering), ¹H NMR relaxation and ThT fluorescence. A minimal reaction model that relates the initial Cu^II binding and Aβ folding with downstream aggregation is presented. We demonstrate that a highly aggregation prone Aβ ? Cu^II species is formed on the sub‐second timescale at mildly acidic pH. This observation might be central to the molecular origin of the known detrimental effect of acidosis in Alzheimer's disease.     

Structurally Diverse Polyamines: Solid‐Phase Synthesis and Interaction with DNA

A versatile solid‐phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher‐order structures of DNA.