Intein-Mediated Construction of a Library of Fluorescent Rab GTPase Probes

Wu, Yao-Wen, Goody, Roger S and Alexandrov, Kirill (2011) Intein-Mediated Construction of a Library of Fluorescent Rab GTPase Probes. Chembiochem, 12 18: 2813-2821. doi:10.1002/cbic.201100377


Author Wu, Yao-Wen
Goody, Roger S
Alexandrov, Kirill
Title Intein-Mediated Construction of a Library of Fluorescent Rab GTPase Probes
Journal name Chembiochem   Check publisher's open access policy
ISSN 1439-4227
1439-7633
Publication date 2011-12
Sub-type Article (original research)
DOI 10.1002/cbic.201100377
Volume 12
Issue 18
Start page 2813
End page 2821
Total pages 9
Place of publication Weinheim, Germany
Publisher Wiley - V C H Verlag GmbH & Co. KGaA
Collection year 2012
Language eng
Formatted abstract
Rab GTPases play a key role in the regulation of membrane trafficking. Post-translational geranylgeranylation is critical for their biological activity and is conferred by Rab geranylgeranyl transferease (RabGGTase), together with an accessory factor, Rab escort protein (REP). Mechanistic studies of Rab prenylation and identification of RabGGTase inhibitors require sensitive reporters of Rab prenylation. In the present work, a combination of protein engineering and expressed protein ligation was used to construct a library of semisynthetic Rab7 fluorescent conjugates. In order to avoid synthesis of a large number of fluorescently labeled peptides, we developed a strategy that combined thiol-reactive dye-labeling of cysteine with in vitro protein ligation. Application of this strategy required optimization of labeling and ligation conditions to promote thiol labeling and disfavor intramolecular cyclization. Using this approach, we constructed 46 fluorescent sensors with different spectral properties that reported on the interaction of Rab7 with RabGGTase, REP-1, and the overall prenylation reaction. Two constructs, Rab7Δ3CCK(NBD) and Rab7Δ2SCCC–dans, displayed 2.5- and 1.5-fold increase in fluorescence, respectively, upon prenylation. Moreover, dansyl-, NBD (4-nitro-benzofurazan)-, I-BA-, and I-SO-labeled Rab7 conjugates exhibited two- to tenfold change in fluorescence upon binding to REP or RabGGTase. These fluorescent sensors allowed us to monitor Rab prenylation in real time and to investigate the assembly of Rab–REP binary and Rab–REP–RabGGTase ternary complexes.
Keyword Fluorescent probes
Inteins
Protein ligation
Protein modifications
Protein prenylation
Geranylgeranyl Transferase
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
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