Influence of Helix Formation on<i>cis/trans</i>Isomerism of Xaa-Pro Bonds Flanking the Helical Segment

Meyer, Sylke; Drewello, Mario; Fischer, Gunter

doi:10.1515/bchm3.1996.377.7-8.489

Cited by 18 publications

(6 citation statements)

References 18 publications

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“…The effect is most striking by going from 10 to 20% TFE in buffer ground electrolyte. This is in good agreement with earlier findings from CD measurements in the case of sCT, where an increase in helicity from 5 to 50% has been observed by changing the TFE content from 10 to 20% [16]. The results of our study make it possible to investigate whether or not the separated conformers exhibit differences in their binding interaction to the corresponding calcitonin receptor, of which cloning was previously reported [22, 231. …”

Section: F Thunecke and G Fischersupporting

confidence: 92%

“…The effect, however, is much smaller in aqueous mixtures containing low amounts of organic solvents. For sCT, Meyer et al [16] have reported a threefold increase in 18% TFE compared to pure water. For the dipeptide Phe-Pro, a twofold increase in water containing 25% methanol was measured [19].…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, structure-inducing effects have also been reported for the peptides under investigation. From CD studies a helix formation induced by TFE and methanol has been published[15,16, 211. An NMR analysis detected structure indications for sCT in 0.9 M SDS[14].…”

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confidence: 99%

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Separation of cis/trans conformers of human and salmon calcitonin by low temperature capillary electrophoresis

Thunecke¹,

Fischer²

1998

Electrophoresis

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Conformer-specific recognition of peptides and proteins has often been proved with the aid of indirect methods. Here we provide an analytical approach for a direct investigation of separated isomers. Cis/trans conformers of the peptide hormones human (hCT) and salmon (sCT) calcitonin exhibit different migration properties in capillary zone electrophoresis at subambient temperatures. Calcitonin consists of 32 amino acids with two proline residues incorporated. It is the longest unstructured peptide for which a conformer separation by capillary electrophoresis has yet been achieved. Lowering the temperature yielded a splitting into two and three peaks for sCT and hCT, respectively, in acidic buffer. The peak ratios of 66:34 for sCT and 71:23 for hCT are in good agreement with the conformer distribution previously reported from nuclear magnetic resonance (NMR) studies. The addition of different organic modifiers (5-20% v/v) to the running buffer does not improve the separation. The observed merging of conformer peaks in buffer containing 20% v/v 2,2,2-trifluoroethanol (TFE) is attributed to structure formation.

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Section: F Thunecke and G Fischersupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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Separation of cis/trans conformers of human and salmon calcitonin by low temperature capillary electrophoresis

Thunecke¹,

Fischer²

1998

Electrophoresis

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“…This could be a consequence of the genomic integration site and/or the high copy-number of the plasmid pSK257 nfap paf_signal in the Penicillium genome. High gene dosage was shown to possibly hamper the transcription or translation machinery of microbial cell factories [36, 37]. However, protein-specific determinants, such as the signal sequence, may also modulate heterologous protein production, processing and secretion in the Penicillium sp.…”

Section: Discussionmentioning

confidence: 99%

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

et al. 2016

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BackgroundSmall, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses.ResultsThe APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays.ConclusionThis study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure–function analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0586-4) contains supplementary material, which is available to authorized users.

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“…Typically, trypsin cleaves after arginine and lysine, while endoproteinase Arg-C cleaves after arginine only (32). Under rare circumstances, however, Arg-C has been shown to cleave after lysine also (46,47). With synthetic substrates, Arg-C exhibits a specificity for arginine that is 100-1000 times greater than that for lysine (48).…”

Section: Discussionmentioning

confidence: 99%

A Bactericidal Monoclonal Antibody Elicits a Change in Its Antigen, OspB of Borrelia burgdorferi, That Can Be Detected by Limited Proteolysis

Katona

Ayalew

Coleman

et al. 2000

The Journal of Immunology

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mAb CB2, directed against outer surface protein B (OspB), causes bacteriolysis of Borrelia burgdorferi in the absence of complement. How this happens is unknown. We examined the effect of mAb binding on OspB tertiary structure by using limited proteolysis to probe changes in protein conformation. Truncated OspB (tOspB) that lacked N-terminal lipid was cleaved by four enzymes: trypsin, endoproteinase Arg-C, endoproteinase Asp-N, and endoproteinase Glu-C. CB2 affected the cleavage by trypsin and Arg-C, but not by AspN or Glu-C. None of the enzymes cleaved CB2 under these conditions. Both trypsin and Arg-C cleaved tOspB near the N-terminus; CB2 slowed the rate of cleavage, but did not affect the identity of the sites cleaved. Irrelevant mAb had no effect, indicating that the effect was specific. CB2 was active against tOspB of strain B31, but not against tOspB of strain BEP4, to which it does not bind, suggesting that binding was required to elicit the effect on cleavage. With trypsin, CB2 showed a maximal effect at 8 mol of tOspB to 1 mol of mAb. At this ratio, not enough CB2 was present to bind all the tOspB; therefore, either CB2 shows turnover or CB2 acts by binding tOspB and effecting a change in this tOspB such that it, in turn, propagates the effect in other molecules of tOspB. Regardless of the mechanism, these data show that CB2 elicits a change in tOspB that can be measured by its reduced susceptibility to protease cleavage.

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Influence of Helix Formation oncis/transIsomerism of Xaa-Pro Bonds Flanking the Helical Segment

Cited by 18 publications

References 18 publications

Separation of cis/trans conformers of human and salmon calcitonin by low temperature capillary electrophoresis

Separation of cis/trans conformers of human and salmon calcitonin by low temperature capillary electrophoresis

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

A Bactericidal Monoclonal Antibody Elicits a Change in Its Antigen, OspB of Borrelia burgdorferi, That Can Be Detected by Limited Proteolysis

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