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

Area of Science:

• Biomolecular NMR Spectroscopy
• Macromolecular Alignment
• Nanomaterials Science

Background:

• Cellulose nanocrystals (CNCs) are inexpensive, non-toxic, and robust alignment media.
• CNCs form liquid crystals in aqueous solutions, enabling macromolecular alignment for NMR.
• Their application as an alignment medium in NMR is underexplored despite advantages.

Purpose of the Study:

• To re-examine the utility of CNCs as an alignment medium for biomolecular NMR.
• To investigate the effects of concentration, ionic strength, and temperature on CNC-mediated molecular alignment.
• To develop a general procedure for preparing aligned samples using CNCs.

Main Methods:

• Analysis of CNCs' effects on molecular alignment under varying conditions (concentration, ionic strength, temperature).
• Testing CNCs' stability across a broad temperature range (10-70°C) and pH range (2.5-8.0).
• Comparison of CNC alignment with a standard medium (Pf1 bacteriophage) using the SH3 domain of Fyn tyrosine kinase.

Main Results:

• Stable molecular alignment was achieved with CNCs over wide temperature and pH ranges.
• CNC suspensions demonstrated high sensitivity to biological buffer concentrations, requiring careful consideration.
• CNCs produced an alignment frame comparable but opposite in magnitude to the Pf1 bacteriophage medium.

Conclusions:

• CNCs are a robust and versatile alignment medium for biomolecular NMR, suitable for thermophilic proteins and acid-stabilized molecules.
• The sensitivity of CNCs to buffer concentration necessitates specific protocols for NMR sample preparation.
• CNCs offer a valuable alternative alignment medium, complementing existing methods like Pf1 bacteriophage.