NMR spectroscopic filtration of polypeptides and proteins in complex mixtures

Journal of Biomolecular NMR
Senapathy RajagopalanSilvia Cavagnero

Abstract

Due to the inherent complexity of the natural biological environment, most studies on polypeptides, proteins and nucleic acids have so far been performed in vitro, away from physiologically relevant conditions. Nuclear magnetic resonance is an ideal technique to extend the in vitro analysis of simple model systems to the more complex biological context. This work shows how diffusion-based spectroscopic selection can be combined with isotopic labeling to tackle and optimize the NMR analysis of specific macromolecules in multicomponent mixtures. Typical media include cell-free systems containing overexpressed proteins, lysates and proteolytic mixtures. We present a few variants of diffusion-edited HSQC pulse sequences for the selective spectroscopic detection of protein and polypeptide resonances within complex mixtures containing undesired species of smaller molecular weight. Due to diffusion-based filtering, peak intensities of fast diffusing small molecules are attenuated more than peaks due to large molecules. The basic sequence, denoted as PFGSTE-HSQC, combines translational diffusion-ordering with two dimensional heteronuclear single quantum correlation spectroscopy. The GCSTE-HSQC and BPPSTE-HSQC sequences include bipolar ...Continue Reading

Citations

Aug 25, 2010·Journal of Biomolecular NMR·Julianne L Kitevski-LeBlancR Scott Prosser
May 10, 2011·Journal of the American Chemical Society·Jung Ho LeeSilvia Cavagnero
May 26, 2009·The Journal of Physical Chemistry. B·Ashok Sekhar, Silvia Cavagnero
May 16, 2007·Journal of Structural Biology·Philipp Selenko, Gerhard Wagner
Oct 3, 2013·Progress in Nuclear Magnetic Resonance Spectroscopy·Christopher A WaudbyJohn Christodoulou
Apr 7, 2010·Journal of Molecular Biology·Stacy-Anne MorganG Andrew Woolley
Jun 15, 2012·Journal of the American Chemical Society·Christopher A WaudbyJohn Christodoulou
Nov 30, 2018·Journal of Biomolecular NMR·Luke W ArbogastJohn P Marino
Mar 19, 2021·Protein Science : a Publication of the Protein Society·Anne R KaplanAndrei T Alexandrescu

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