Protein alignment using cellulose nanocrystals: practical considerations and range of application.

Cellulose nanocrystals (CNCs) form liquid crystals in aqueous solution that confer alignment to macromolecules and permit the measurement of residual dipolar couplings. CNCs possess many attractive features as an alignment medium. They are inexpensive, non-toxic, chemically inert, and robust to denaturants and temperature. Despite these advantages, CNCs are seldom employed as an alignment medium and the range of their applicability has not yet been explored. We have re-examined the use of CNCs in biomolecular NMR by analyzing the effects concentration, ionic strength, and temperature on molecular alignment. Stable alignment was obtained over wide ranges of temperature (10-70 degrees C) and pH (2.5-8.0), which makes CNCs potentially very useful in studies of thermophilic proteins and acid-stabilized molecules. Notably, we find that CNC suspensions are very sensitive to the concentrations of biological buffers, which must be taken into account when they are used in NMR analyses. These results have led us to develop a general procedure for preparing aligned samples with CNCs. Using the SH3 domain from the Fyn tyrosine kinase as a model system, we find that CNCs produce an alignment frame collinear with that of the commonly used Pf1 bacteriophage alignment medium, but of opposite magnitude.