Affinity maturation of a portable Fab-RNA module for chaperone-assisted RNA crystallography

Nucleic Acids Research
Deepak KoiralaJoseph A Piccirilli

Abstract

Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3-6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5'-GNGACCC-3' consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab-RNA module may facilitate its use as an affini...Continue Reading

References

Jan 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·T A Kunkel
May 1, 1994·Trends in Biotechnology·T Clackson, J A Wells
Jan 1, 1994·Methods in Molecular Biology·M K Trower
Dec 11, 1999·Nucleic Acids Research·G Johnson, T T Wu
Dec 11, 1999·Nucleic Acids Research·H M BermanP E Bourne
Mar 13, 2001·Methods : a Companion to Methods in Enzymology·C KaoM Zheng
Jun 26, 2003·Nucleic Acids Research·Michael Zuker
Jun 3, 2004·Genome Research·Gavin E CrooksSteven E Brenner
Jan 19, 2007·Nature Reviews. Genetics·Kevin Chen, Nikolaus Rajewsky
Aug 8, 2007·Journal of Molecular Biology·Evgeny Krissinel, Kim Henrick
Dec 29, 2007·Proceedings of the National Academy of Sciences of the United States of America·Jing-Dong YeJoseph A Piccirilli
Feb 26, 2009·Cell·Chris P PontingWolf Reik
May 22, 2009·Nucleic Acids Research·Timothy L BaileyWilliam S Noble
Jan 22, 2010·Current Opinion in Structural Biology·Andrew J Newman, Kiyoshi Nagai
Mar 5, 2010·Current Protocols in Nucleic Acid Chemistry·Bradley HallAndrew D Ellington
May 5, 2010·Nucleic Acids Research·Mickael GoujonRodrigo Lopez
Dec 15, 2010·Nature Structural & Molecular Biology·Yelena KoldobskayaJoseph A Piccirilli
Sep 20, 2011·Molecular Cell·Ronald R Breaker
Dec 26, 2012·Bioarchitecture·Irina V NovikovaKarissa Y Sanbonmatsu
Jul 31, 2013·Nature·Jinwei Zhang, Adrian R Ferré-D'Amaré
Mar 13, 2014·Current Opinion in Structural Biology·Jinwei Zhang, Adrian R Ferré-D'Amaré
Apr 20, 2014·Science·Erich G ChapmanJeffrey S Kieft
Jun 24, 2014·Nature Chemical Biology·Hao HuangJoseph A Piccirilli
Oct 13, 2014·Nature Chemical Biology·Richard I McDonaldDavid R Liu
Jan 25, 2016·Current Opinion in Structural Biology·Thi Hoang Duong NguyenKiyoshi Nagai
Sep 7, 2016·Journal of Molecular Biology·Yaming ShaoJoseph A Piccirilli
Mar 28, 2017·Current Opinion in Structural Biology·Dominika ElmlundHans Elmlund
Apr 26, 2017·Trends in Biochemical Sciences·Chen Zhao, Anna Marie Pyle
Aug 30, 2017·Current Opinion in Structural Biology·Ottilie von LoeffelholzBruno P Klaholz

❮ Previous
Next ❯

Citations

Aug 14, 2018·Angewandte Chemie·Saskia Neubacher, Sven Hennig
Jan 28, 2020·Journal of Materials Chemistry. B, Materials for Biology and Medicine·Puchakayala SwethaJian-Hui Jiang
Aug 20, 2019·Quarterly Reviews of Biophysics·Robert J Trachman, Adrian R Ferré-D'Amaré
Jul 10, 2019·Methods : a Companion to Methods in Enzymology·Sizhuo ChenKe Liu

❮ Previous
Next ❯

Methods Mentioned

BETA
X-ray
nuclear magnetic resonance
phage display
in vitro transcription
electrophoresis
PCR
filter binding
filter
foot-printing
immunoprecipitation

Software Mentioned

Phenix
Clustal Omega
Multiple Em for Motif Elicitation ( MEME )
Phaser
PyMOL
SNRK
WebLogo
COOT
PDBePISA
CorelDraw

Related Concepts

Related Feeds

Bacteriophage: Phage Therapy

Phage therapy uses bacterial viruses (bacteriophages) to treat bacterial infections and is widely being recognized as an alternative to antibiotics. Here is the latest research.