Abstract
The protein unfolding is highly related with the maintenance of protein structures and functions in certain content. Therefore, to study protein early-stage unfolding process would be critical in understanding the diseases induced by protein unfolding. Here, low Urea concentration (< 1.5 M) is used to destabilize Ubiquitin and derive a structure representing the early-unfolding state. We used a series of NMR methods to detect protein backbone dynamics at the early-unfolding state. In the first examination, we measured T1, T2, NOE of individual residues and employed ModelFree method to analyze the dynamic parameters. Low concentration Urea led reduced S2 and increased number of residues with conformational exchange (Rex). Meanwhile, we also use a modern field-cycling NMR (FCNMR) method to detect the longitudinal relaxation rates (T1) of the individual Ubiquitin backbone amides under the magnetic field strengths from 20 Telsa (850 MHz) to ~1 Telsa (42.5 MHz). The field-dependent relaxations precisely reported backbone internal motion with wider time scale (nanosecond to picosecond). The result well corresponded the result derived from T1, T2, NOE measurements and ModelFree analysis. We also performed secondary structure prediction and hydrogen bond detection by NMR and the results showed less significant change in structure. In summary, NMR approaches including FCNMR technology could report a wider range of time-scale for molecular dynamics, providing a more complete insight and dynamic description for studying protein early-stage unfolding process.