# Measuring 15N T1 and T2 relaxation times (Varian)

### Experimental Setup

Run the `HTP_t1t2_setup` script to setup the following experiments:

- 2D [15N, 1H] HSQC, ~1.5h for SN analysis
`NHonly='n' C13refoc='y' ni=256 f1180='n' phase=1,2 nt=8 ss=32`

- 1D 15N T1, ~30 min
`ni=1 phase=1 T1='y' f1180='n' NHonly='n' C13refoc='n' ss=256 nt=128``relaxT=0.1, 0.2, 0.3, 0.4, 0.7, 1.0, 1.5, 2.0`

- 1D 15N T2, ~30 min
`ni=1 phase=1 T2='y' f1180='n' NHonly='n' C13refoc='n' ss=256 nt=128``relaxT=0.01,0.03,0.05,0.07,0.09,0.11,0.13,0.15,0.17 maxrelaxT=0.17`

Key points to consider:

- For most proteins it is recommended to run T1 and T2 experiments for at least 30 min each to achieve adequate S/N on a room-temperature probe. Short measurement times may lead to underestimated tc values. With cryogenic probes or very concentrated (> 1 mM) samples the minimum measurement time may be smaller.
- Short
`d1`delays (~1 s) may lead to incorrect integral for the first T1 point. However, long d1 values may lead to a large residual water line and non-uniform baseline, especially on a cryoprobe. See what works best for the particular sample and spectrometer. `relaxT`must be given as a multiple of 10 ms for T1 and**odd**multiple of 10 ms for T2.`maxrelaxT`is not used in T1 measurements.- Avoid sampling T2 points beyond 250 ms - it may cause excessive sample heating.
- Intermediate tc values (between monomer and dimer) may indicate transient dimerization. Dilution studies are then required.
- The tc value is calculated under assumption of isotropic tumbling. For example, if a protein consists of long parallel a-helices the reported tc will indicate a larger molecular weight.

### Calculating rotational correlation time

- Process the data in Buffalo.VNMR with
`wft`and adjust the phase for pure absorption. - Invoke
`dc`to correct baseline shift and slope. Expand the spectrum first for better results. - Invoke
`dscale`and`setref`to make sure the ppm scale is correct - required for the`tc`macro. - Display all spectra with
`dssh`and check that- baseline is flat and uniform in all 1D spectra.
- Spectral intensity follows an exponential decay.

- Run
`tc([T1exp, T2exp, [ppm1, ppm2]])`, where the first two arguments are T1 and T2 experiment numbers, and the last two optional arguments specify the integration range in ppm. By default, integration is performed over the range between 10.5 ppm and 8.5 ppm to exclude signals from side-chain CONH2 groups (6 - 7 ppm) and unfolded segments (8 ppm). If called with no arguments it will prompt for experiment numbers and use the default integration range.

The `tc` macro requires `t1a`, `t2a` and `intav` macros. The `intav` macro performs integration and stores the integral values in the `fp.out` file. `t1a` and `t2a` macros extract T_{1} and T_{2} relaxation times from the exponential fitting to the data in `fp.out`. Manual phase and drift correction (`dc`) of 1D spectra should be performed first to get accurate results.

Rotational correlation time τ_{c} is then calculated as

- <math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,

where ν_{N} is the ^{15}N resonance frequency.

The reported error range is from simple error propagation of the exponential fit error and is rather crude. Strictly speaking, such simple error propagation does not apply here, because the expected error distribution for τ_{c} is asymmetric.

To install tc macro:

- Download tc_macro.tar into the user maclib directory (
`~/vnmrsys/maclib/`) - Unpack it with
`tar xvf tc_macro.tar`

-- AlexEletski - 03 Mar 2008