Methods to determine slow diffusion coefficients of biomolecules. Applications to Engrailed 2, a partially disordered protein
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We present new NMR methods to measure slow translational diffusion coefficients of biomolecules. Like the heteronuclear stimulated echo experiment (XSTE), these new methods rely on the storage of information about spatial localization during the diffusion delay as longitudinal polarization of nuclei with long T1 such as nitrogen-15. The new BEST-XSTE sequence combines features of Band-selective Excitation Short-Transient (BEST) and XSTE methods. By avoiding the saturation of all protons except those of amide groups, one can increase the sensitivity by 45% in small proteins. The new experiment which combines band-Selective Optimized Flip-Angle Short-Transient with XSTE (SOFAST-XSTE) offers an alternative when very short recovery delays are desired. A modification of the HSQC-edited version of the XSTE experiment offers enhanced sensitivity and access to higher resolution in the indirect dimension. These new methods have been applied to detect changes in diffusion coefficients due to dimerization or proteolysis of Engrailed 2, a partially disordered protein.
KeywordsTranslational diffusion Heteronuclear stimulated echoes Proteolysis Dimerization Disulfide bonds Partially disordered proteins
Gilles Clodic and Gérard Bolbach are acknowledged for MALDI-TOF spectra acquisition and analysis. We thank Pau Bernado (IRB, Barcelona) and Damien Baigl (Ecole Normale Supérieure, Paris) for fruitful discussions.
- Augé S, Schmit PO, Crutchfield CA, Islam MT, Harris DJ, Durand E, Clemancey M, Quoineaud AA, Lancelin JM, Prigent Y, Taulelle F, Delsuc MA (2009) NMR measure of translational diffusion and fractal dimension. Application to molecular mass measurement. J Phys Chem B 113:1914–1918. doi: 10.1021/jp8094424 CrossRefGoogle Scholar
- Augustyniak R, Balayssac S, Ferrage F, Bodenhausen G, Lequin O (2011) 1H, 13C and 15N resonance assignment of a 114-residue fragment of Engrailed 2 homeoprotein, a partially disordered protein. Biomol NMR Assign. doi: 10.1007/s12104-011-9306-5
- Cantor CR, Schimmel PR (1980) Biophysical chemistry, part II: techniques for the study of Biological Structure and Function. W.H. Freeman, San FranciscoGoogle Scholar
- Chang PT (2000) Chapter 21.4 diffusion. In: Physical chemistry for the chemical and biological sciences, 2nd edn, University Science Books, Sausalito, CA, pp 876–883Google Scholar
- Choy WY, Mulder FAA, Crowhurst KA, Muhandiram DR, Millett IS, Doniach S, Forman-Kay JD, Kay LE (2002) Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques. J Mol Biol 316:101–112. doi: 10.1006/jmbi.2001.5328 CrossRefGoogle Scholar
- Danielsson J, Jarvet J, Damberg P, Graslund A (2002) Translational diffusion measured by PFG-NMR on full length and fragments of the Alzheimer Aβ(1–40) peptide. Determination of hydrodynamic radii of random coil peptides of varying length. Magn Reson Chem 40:S89–S97. doi: 10.1002/mrc.1132 CrossRefGoogle Scholar
- Danielsson J, Liljedahl L, Barany-Wallje E, Sønderby P, Kristensen LH, Martinez-Yamout MA, Dyson HJ, Wright PE, Poulsen FM, Maler L, Graslund A, Kragelund BB (2008) The intrinsically disordered RNR inhibitor Sml1 is a dynamic dimer. Biochemistry 47:13428–13437. doi: 10.1021/bi801040b CrossRefGoogle Scholar
- Orekhov VY, Korzhnev DM, Pervushin KV, Hoffman E, Arseniev AS (1999) Sampling of protein dynamics in nanosecond time scale by 15 N NMR relaxation and self-diffusion measurements. J Biomol Struct Dynam 17:157–174Google Scholar
- Price WS (1998) Pulsed-field gradient nuclear magnetic resonance as tool for studying translational diffusion: part II. Experimental aspects. Concepts Magn Reson 10:197–237. doi: 10.1002/(SICI)1099-0534(1998)10:4<197::AID-CMR1>3.0.CO;2-S CrossRefGoogle Scholar