BEGIN:VCALENDAR VERSION:2.0 PRODID:-//jEvents 2.0 for Joomla//EN CALSCALE:GREGORIAN METHOD:PUBLISH BEGIN:VEVENT UID:6fde063aefd123bf2086bf0f9122a481 CATEGORIES:Colloquium CREATED:20210914T164757 SUMMARY:Professor Carl Denard, University of Florida DESCRIPTION:
Deciphering and Engineering the Substrate Specificity of Protein-Modifying Enzymes
Protein-modifying enzym es (PMEs) are ubiquitous in biology, playing significant roles in initiatin g, regulating, and terminating cellular processes. The diversity and breadt h of their substrate preference have been harnessed for a variety of applic ations, including protease therapeutics, protein purification, mass spectro metry-based proteomics, targeted therapeutics, and site-specific bioconjuga tions. Furthermore, with advances in protein engineering and synthetic biol ogy, they are now being utilized for studying protein-protein interactions, imaging newly synthesized proteins, and performing logic operations inside cells. To successfully repurpose PMEs towards these applications, it is of ten necessary to engineer their catalytic properties and to profile their s ubstrate specificities. In searching for desired activities in a large pool of variants, a high-throughput screening or selection system should ideall y exhibit a broad operational range (dose-response or sensitivity over a la rge variation in input), and a high dynamic range (signal-to-noise ratio).< /p>
Here we describe YESS 2.0, a highly modu lar and customizable yeast endoplasmic sequestration screening system suita ble for engineering and profiling the specificity of protein-modifying enzy mes. By incorporating features to modulate gene transcription, as well as s ubstrate and enzyme spatial sequestration within a versatile and seamless a ssembly method, YESS 2.0 achieves broad operational and dynamic range. To s howcase YESS 2.0, we evolve a TEV protease variant (eTEV) with an 8-fold hi gher catalytic efficiency to obtain the fastest TEV protease variant to dat e. Due to the unique features of our system, this phenotype is strictly att ributable to an increase in turnover number (kcat). Second, we use YESS 2.0 coupled with NextGen Sequencing to profile the substrate specificity of in sulin-degrading enzyme (IDE) and discover IDE substrates with highly increa sed activity compared to the prototypical insulin peptide. Lastly, we show for the first time that YESS 2.0 supports calcium-independent sortase-media ted ligations (SML) and confirm that residues directly C-terminal of the pe ntapeptide motif heavily influence the rate of SML. This state-of-the-art p latform offers unmatched versatility in profiling and engineering the speci ficity of protein-modifying enzymes and should enable even more ambitious f uture undertakings.
Hosted by Professor Sagar Khare
For Zoom meeting information, please contact Loretta Lupo @
Deciphering and Engineering the Substrate Specific ity of Protein-Modifying Enzymes
Protein-modifying enzymes (PMEs) are ubiquitous in biology, playing signi ficant roles in initiating, regulating, and terminating cellular processes. The diversity and breadth of their substrate preference have been harnesse d for a variety of applications, including protease therapeutics, protein p urification, mass spectrometry-based proteomics, targeted therapeutics, and site-specific bioconjugations. Furthermore, with advances in protein engin eering and synthetic biology, they are now being utilized for studying prot ein-protein interactions, imaging newly synthesized proteins, and performin g logic operations inside cells. To successfully repurpose PMEs towards the se applications, it is often necessary to engineer their catalytic properti es and to profile their substrate specificities. In searching for desired a ctivities in a large pool of variants, a high-throughput screening or selec tion system should ideally exhibit a broad operational range (dose-response or sensitivity over a large variation in input), and a high dynamic range (signal-to-noise ratio).
Here we describ e YESS 2.0, a highly modular and customizable yeast endoplasmic sequestrati on screening system suitable for engineering and profiling the specificity of protein-modifying enzymes. By incorporating features to modulate gene tr anscription, as well as substrate and enzyme spatial sequestration within a versatile and seamless assembly method, YESS 2.0 achieves broad operationa l and dynamic range. To showcase YESS 2.0, we evolve a TEV protease variant (eTEV) with an 8-fold higher catalytic efficiency to obtain the fastest TE V protease variant to date. Due to the unique features of our system, this phenotype is strictly attributable to an increase in turnover number (kcat) . Second, we use YESS 2.0 coupled with NextGen Sequencing to profile the su bstrate specificity of insulin-degrading enzyme (IDE) and discover IDE subs trates with highly increased activity compared to the prototypical insulin peptide. Lastly, we show for the first time that YESS 2.0 supports calcium- independent sortase-mediated ligations (SML) and confirm that residues dire ctly C-terminal of the pentapeptide motif heavily influence the rate of SML . This state-of-the-art platform offers unmatched versatility in profiling and engineering the specificity of protein-modifying enzymes and should ena ble even more ambitious future undertakings.
Hosted by Profess or Sagar Khare
For Zoom meeting information, please contact Lo
retta Lupo @