Translational diffusion of macromolecular assemblies measured using transverse-relaxation-optimized pulsed field gradient NMR.

Abstract

In structural biology, pulsed field gradient (PFG) NMR spectroscopy for the characterization of size and hydrodynamic parameters of macromolecular solutes has the advantage over other techniques that the measurements can be recorded with identical solution conditions as used for NMR structure determination or for crystallization trials. This paper describes two transverse-relaxation-optimized (TRO) (15)N-filtered PFG stimulated-echo (STE) experiments for studies of macromolecular translational diffusion in solution, (1)H-TRO-STE and (15)N-TRO-STE, which include CRINEPT and TROSY elements. Measurements with mixed micelles of the Escherichia coli outer membrane protein X (OmpX) and the detergent Fos-10 were used for a systematic comparison of (1)H-TRO-STE and (15)N-TRO-STE with conventional (15)N-filtered STE experimental schemes. The results provide an extended platform for evaluating the NMR experiments available for diffusion measurements in structural biology projects involving molecular particles with different size ranges. An initial application of the (15)N-TRO-STE experiment with very long diffusion delays showed that the tedradecamer structure of the 800 kDa Thermus thermophilus chaperonin GroEL is preserved in aqueous solution over the temperature range 25-60 °C.