Motions at the milli / microsecond time scale are considered important for biological functioning. Enzymes open and close the active site cleft, molecules change their conformation, the interaction interface between molecules dynamically adepts. Currently there are several publications from several groups at visible places that highlight these dynamics.

Ishima, R., Freedberg, D.I., Wang, Y.X., Louis, J.M. & Torchia, D.A. (1999) Structure Fold. Des. 7, 1047-1055 .
Hodsdon, M.E. & Cistola, D.P. (1997) Biochemistry 36, 2278-2290 .
Feher, V.A. & Cavanagh, J. (1999) Nature 400, 289-293 .
Volkman, B.F., Lipson, D., Wemmer, D.E. & Kern, D. (2001) Science 291, 2429-2433.
Wang, L., Pang, Y., Holder, T., Brender, J.R., Kurochkin, A, Zuiderweg, E.R.P. Functional Dynamics in the active site of the ribonuclease Binase, Proc. Natl. Acad. Sci. USA, 2001, in press
Stevens, S.Y., Sanker, S., Kent, C. Zuiderweg, E.R.P. Delineation of the allosteric mechanism for a cytidylyltransferase  exhibiting negative cooperativity, Nature Struct. Biol., in press
 

The conventional way to obtain these important milii/micro second dynamical events is by recording 15N R1, R2 and NOE data, and the Modelfree methodology to extract the Rex rates. It should be realized, however, that most research teams run the R2 experiments using CPMG sequences, suppressing the exchange broadening! Collectively as NMR spectroscopists, we may have suppressed much important information with this practice.

A simple protocol to detect exchange broadening is to compare a R2 sequence in which the exchange is optimally suppressed (R2-CPMG or T1rho) with one in which it is least suppressed. If desired, the Rex rates can be quantitatively converted to exchange rates using one of the methodologies developed in Dr. A. Palmer's lab.

Below we show two sequences that we have used to carry out the detection of Rex effects. The R2 CPMG sequence is standard, but is intentionally primitive: water is kept saturated during the sequence, as we want to compare with the R2 sequence. No sensitivity enhancements or Trosy  here, because the sequence should be usable for proteins with much exchange broadening - thus the 15N transverse time around the relaxation period needs to be as short as possible for best sensitivity.
The R2 sequence is trivially simple and we used it in our latest two publications of Wang et al (PNAS) and Stevens et al (Nat. Str. Biol). This simple sequence covers most grounds:  No anti-phase 15N-1H magnetization occurs during the T2 period because of the proton CPD decoupling, so no need for balancing in and anti-phase relaxation; the proton decoupling takes care of the CSA/DD cross correlations. We have seen no problems with refocusing of the 15N magnetization in the presence of proton decoupling - for safety one can temporarily switch off the CPD decoupling during the 15N refocusing pulse. Potential problems: diffusion during the T2 series is not quenched (but we saw no problem even with a small protein). Potential general benefit: this sequence is less sensitive to r.f. offsets than the  CPMG.

 15N R2 sequence for Varian (but easy to reprogram for Bruker)
 15N CPMG sequence for Varian (but easy to reprogram for Bruker)
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