Design of small volume HX and triple-resonance probes for improved limits of detection in protein NMR experiments

Three- and four-frequency nuclear magnetic-resonance probes have been designed for the study of small amounts of protein. Both "HX" (H-1, X, and H-2 channels) and "triple-resonance" (H-1, N-15, C-13, and H-2) probes were implemented using a single transmit/receive coil and multiple-frequency impedance matching circuits. The coil used was a six-turn solenoid with an observe volume of 15 mul. A variable pitch design was used to improve the B-1 homogeneity of the coil. Two-dimensional HSQC spectra of similar to1 mM single labeled N-15- and double labeled N-15/C-13-proteins were acquired in experimental times of approximately 2h. Triple-resonance capability of the small-volume triple-resonance probe was demonstrated by acquiring three-dimensional HNCO spectra from the same protein samples. In addition to enabling very small quantities of protein to be used, the extremely short pulse widths (H-1 = 4, N-15 = 4, and C-13 = 2 mus) of this particular design result in low power decoupling and wide-bandwidth coverage, an important factor for the ever-higher operating frequencies used for protein NMR studies. (C) 2003 Elsevier Science (USA). All rights reserved.