Measuring translational diffusion of 15 N-enriched biomolecules in complex solutions with a simplified 1 H- 15 N HMQC-filtered BEST sequence.

Pulsed-field gradient nuclear magnetic resonance has seen an increase in applications spanning a broad range of disciplines where molecular translational diffusion properties are of interest. The current study introduces and experimentally evaluates the measurement of translational diffusion coefficients of ¹⁵ N-enriched biomolecules using a ¹ H- ¹⁵ N HMQC-filtered band-selective excitation short transient (BEST) sequence as an alternative to the previously described SOFAST-XSTE sequence. The results demonstrate that accurate translational diffusion coefficients of ¹⁵ N-labelled peptides and proteins can be obtained using this alternative ¹ H- ¹⁵ N HMQC-filtered BEST sequence which is implementable on NMR spectrometers equipped with probes fitted with a single-axis field gradient, including most cryoprobes dedicated to bio-NMR. The sequence is of potential use for direct quantification of protein or peptide translational diffusion within complex systems, such as in mixtures of macromolecules, crowded solutions, membrane-mimicking media and in bicontinuous cubic phases, where conventional sequences may not be readily applicable due to the presence of intense signals arising from sources other than the protein or peptide under investigation.