The described TROSY-based experiments for investigating backbone dynamics of proteins make it possible to elucidate internal motions in large proteins via measurements of T 1 , T 2 , and NOE of backbone 15 N nuclei. In our proposed sequences, the INEPT sequence is eliminated and the PEP sequence is replaced by the ST2-PT sequence from the HSQC-based experiments. This has the benefit of shortening the pulse sequences by 5.4 ms (=1/2J) and results in an increase in the intrinsic sensitivity of the proposed TROSY-based experiments. The TROSY-based experiments are on average of 13% more sensitive than the corresponding HSQC-based experiments on a uniformly 15 N-labeled Xenopus laevis calcium-bound calmodulin sample on a 750-MHz spectrometer at 5°C. The amide proton linewidths of the TROSY-based experiments are 2-13 Hz narrower than those of the HSQC experiments. More sensitivity gain and higher resolution are expected if the protein sample is deuterated.
Bibliographical noteFunding Information:
This work is supported by grants from the Research Grant Council of Hong Kong (HKUST6197/97M, HKUST6038/98M, and HKUST6199/99M). The Hong Kong Biotechnology Research Institute is acknowledged for the purchase of the 750-MHz NMR spectrometer. LKN acknowledges support from the National Science Foundation (MCB-9808727) and the National Institutes of Health (1 R01 CA77478-01).
- Protein dynamics