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Updated: May 18, 2026

In Vivo Proximity Biotinylation for Protein Interaction Studies in Paramecium tetraurelia
Published on: September 12, 2025
Expansion of protein farnesyltransferase specificity using "tunable" active site interactions: development of
James L Hougland1, Soumyashree A Gangopadhyay, Carol A Fierke
1Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA. hougland@syr.edu
Abstract:
Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be "tuned" using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein.
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