SummaryFunctional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes but structural studies on this part of the proteome are limited. In this study we undertook such an approach to analyse photosynthetic thylakoid membranes isolated from wild-type and mutant strains of Chlamydomonas reinhardtii. Thylakoid membrane proteins were separated by high-resolution two-dimensional gel electrophoresis (2-DE) and analysed by immunoblotting and mass spectrometry for the presence of membrane-spanning proteins. Our data show that light-harvesting complex proteins (LHCP), that cross the membrane with three transmembrane domains, can be separated using this method. We have identi®ed more than 30 different LHCP spots on our gels. Mass spectrometric analysis of 2-DE separated Lhcb1 indicates that this major LHCII protein can associate with the thylakoid membrane with part of its putative transit sequence. Separation of isolated photosystem I (PSI) complexes by 2-DE revealed the presence of 18 LHCI protein spots. The use of two peptide-speci®c antibodies directed against LHCI subunits supports the interpretation that some of these spots represent products arising from differential processing and post-translational modi®cations. In addition our data indicate that the reaction centre subunit of PSI, PsaA, that possesses 11 transmembrane domains, can be separated by 2-DE. Comparison between 2-DE maps from thylakoid membrane proteins isolated from a PSI-de®cient (Dycf4) and a crd1 mutant, which is conditionally reduced in PSI and LHCI under copper-de®ciency, showed the presence of most of the LHCI spots in the former but their absence in the latter. Our data demonstrate that (i) hydrophobic membrane proteins like the LHCPs can be faithfully separated by 2-DE, and (ii) that high-resolution 2-DE facilitates the comparative analysis of membrane protein complexes in wild-type and mutants cells.
The ergot alkaloids, a class of fungal-derived natural products with important biological activities, are derived from a common intermediate, chanoclavine-I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast-based expression platform along with in vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine-I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500 mg L−1, thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.
Synthetic
biology has been heralded as a new bioengineering platform
for the production of bulk and specialty chemicals, drugs, and fuels.
Here, we report for the first time a series of 74 novel compounds
produced using a combinatorial genetics approach in baker’s
yeast. Based on the concept of “coevolution” with target
proteins in an intracellular primary survival assay, the identified,
mostly scaffold-sized (200–350 MW) compounds, which displayed
excellent biological activity, can be considered as prevalidated hits.
Of the molecules found, >75% have not been described previously;
20%
of the compounds exhibit novel scaffolds. Their structural and physicochemical
properties comply with established rules of drug- and fragment-likeness
and exhibit increased structural complexities compared to synthetically
produced fragments. In summary, the synthetic biology approach described
here represents a completely new, complementary strategy for hit and
early lead identification that can be easily integrated into the existing
drug discovery process.
Polymeric receptors prepared by a combination of covalent and noncovalent imprinting show high selectivity for the amino acid sequence of the template (see schematic representation of the binding of the tripeptide Lys‐Trp‐Asp).
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