Measuring 15N T1 and T2 relaxation times (Bruker)

Introduction

The rotational correlation time of a protein in solution is the time for a protein to rotate one radian. In the limit of slow molecular motion (τ_c >> 0.5 ns), the correlation time of a protein is related to the ratio of the longitudinal (T₁) and transverse (T₂) ¹⁵N relaxation times, and nuclear frequency (ν_N) according to Eq. 1, which is derived from Eq. 8 in Kay et al. (Ref. 1) by considering only J(0) and J(ω) spectral densities and neglecting higher frequency terms.         

In practice, global ¹⁵N T₁ and T₂ relaxation times for an unknown protein target can be obtained quickly (ca. 1 h) on a 1.7-mm microcyroprobe using 1D ¹⁵N-edited relaxation experiments (Ref. 2), by fitting integrated signal in the backbone amide ¹H region of the spectrum as a function of delay time to an exponential decay. One then computes the correlation time using Eq. 1, and compares it to a standard curve of τ_c vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size.  As a general rule of thumb, the τ_c of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kiloDaltons. This approach is reliable up to MW ≈ 25 kDa, where accurate measurement of the diminishing ¹⁵N T₂ becomes problematic.

Methods

Acquisition of 1D ¹⁵N T₁ and T₂ on Bruker Spectrometers

On Bruker spectrometers equipped with TopSpin 2.1 we use the hsqct1etf3gpsi3d and hsqct2etf3gpsi3d pulse sequences for the acquisition of ¹⁵N T₁ and T₂ spectra (Ref. 1-3). The 1D version of these experiments are acquired as pseudo 2D spectra, where the ¹H^N dimension is arrayed vs. delay time. We acquire ¹H-detected 1D ¹⁵N relaxation spectra by using the following settings in the acquisition mode:

• set the number of acquisition points in the ¹⁵N dimension, td2 = 1
• set the number of points in the 3rd dimension equal to the number of delay times used in the experiment
• set the acquisition mode in the 3rd dimension to QF
• set NBL equal to the number of delay times used in the experiment; this is very important for the experiment to work properly
• define a vdlist (T₁) or vclist (T₂)
• set d1 to 3 - 5 s (T₁) or 1.5 - 3 s (T₂) for adequate relaxation during the experiment.

Typical vdlist and vclist files are attached.