C – 7995 Practicals 1 – preparation for measurement
A) Shimming
Use sample: chloroform in acetone 1%
For NMR700m, you can use parameter set: exp. 101 in directory /d1/fiala/nmr/lineshape/
Shim the sample as best as you can by combination of methods; verify the result by measuring a
spectrum and evaluating it by humpcal command.
Note: 5 mm probe specification is 10/20 Hz for non-rotating sample.
B) Pulse calibration
Use sample: doped water
Use parameter set: zg
Calibrate 90⁰ on 1
H manually (command paropt) and automatically (pulsecal)
Compare the values obtained at 180⁰, 360⁰, and by pulsecal
C) Temperature calibration
Use sample: methanol 4%
Use parameter set: zg
Calibrate the temperature at 25⁰C and 10⁰C, and calculate the correction parameters for the VT unit.
Use calctemp command or NMR-TempCal.xls spreadsheet to translate the chemical shift difference
into temperature.
Use the following procedure: set the temperature, wait till the temperature stabilizes, measure the
spectrum and evaluate the temperature, wait for 5 min., measure and evaluate again, if the
temperature difference is more than 0.1 K, wait another 5 min., repeat the measurement . . .
C7995 Practicals 2 – 1D NMR spectroscopy
1. NMR spectra in organic solvent
Measure proton spectrum of a simple compound in organic solvent
Use sample: 0.1% Ethylbenzene in CDCl3 (Bruker standard test sample)
Pulse sequence: zg
Parameters: standard parameters – PROTON, change the pulse sequence to zg, adjust spectral width
according to the needs, optimize 90⁰ pulse
13
C spectrum
Use sample: 20% Ethylbenzene in CDCl3 (Bruker standard test sample for 13
C)
Pulse sequence: zgpg
Parameters: standard parameters – C13CPD, change the pulse program to zgpg, adjust spectral width
according to the needs, use prosol pulse length for 13
C pulse and the proton pulse calibration from
the previous step. Measure the spectrum with and without proton decoupling, compare the results.
Note the splitting of the CDCl3 signal (around 77 ppm) by deuterium.
2. NMR spectra in water solution
Measure proton spectrum of DNA dissolved in water using solvent suppression techniques
Use sample: Dickerson-Drew DNA dodecamer d(CGCGAATTCGCG)2 in 90% H2O/10% D2O at 25⁰C
Pulse sequences: zgpr, p3919gp and zggpwg
Parameters: standard parameters (ZGPR, P3919PG, ZGGPWG), adjust the spectral width (imino
protons appear between 12 and 14 ppm, adjust sw and parameter d19 in p3919gp accordingly!),
calibrate proton 90⁰ pulse using pulsecal.
Measure the spectrum using presaturation and WATERGATE techniques, compare the intensities of
the imino signals.
On the same sample of DNA, measure 31
P spectrum
Pulse sequence: zgdc
Parameters: standard parameters – P31CPD, adjust spectral width (phosphate signals appear close to
0 ppm). Use 31
P pulse length from prosol and proton pulse length based on the calibration in the
previous step. Verify the 31
P 90⁰pulse length on the buffer signal.
C7995 Practicals 3 – 2D homonuclear spectroscopy
A) Through-bond correlation experiments
Measure COSY and TOCSY spectra of DNA in D2O at 25⁰C with presaturation of the residual water
signal. Before the 2D, record also 1D spectrum.
Use pulse sequences: zgpr, cosyphpr, dipsi2phpr.
Based on the 1D spectrum, set a suitable spectral width (sw).
Measure the COSY spectrum with 16 scans and 1200 increments.
Measure the TOCSY spectrum with 50 ms mixing time, 8 scans and 600 increments.
Do not forget to scale down the presaturation power by 20 dB compared to water spectra.
B) 2D NOE spectra
Use sample: DNA in 90% H2O/10% D2O at 10⁰C
Measure 2D NOESY spectrum of the sample above using WATERGATE solvent suppression (pulse
sequences noesyfpgpphwg or noesyfpgpph19). To find a suitable spectral width check the spectrum
measured in Practicals 2. Measure the spectrum with 200 ms mixing time, 16 scans and 1024
increments. When optimizing the water suppression measure the first increment with- and without
the flip-back pulse and compare the effect on the imino signals.
Practical 4 – 2D heteronuclear spectroscopy of DNA and isotopically labeled protein sample
Samples:
Ubiquitin, 15
N and 13
C labeled, in 90%H2O/10% D2O
Dickerson-Drew DNA dodecamer d(CGCGAATTCGCG)2 at natural isotopic abundance in D2O
A) 1
H-15
N correlation
Measure 1
H-15
N HSQC spectra using HSQC (pulse sequence sfhsqcf3gpph) and gradient sensitivity
enhanced HSQC (hsqcetf3gpsi2) on the Ubiquitin sample. In the first case, use the spectral width
sufficient to include signals from Arginine sidechains. In the second spectrum, set the spectral width
for the amide region only. In the resulting spectrum, indicate the folded peaks from the Arginine
sidechains. In both cases, use a refocusing carbon pulse to eliminate the effect of carbon coupling in
the indirect dimension (flag LABEL_CN).
For the protein chemical shifts, refer to the attached table or go to BMRB database
http://www.bmrb.wisc.edu/ref_info/
B) 1
H-13
C correlation
On the Ubiquitin sample, measure 1
H-13
C HSQC spectra of the aliphatic region (5 ppm to 75 ppm in
the carbon dimension) using standard (e.g. hsqcetgp, hsqcetgpsi) and constant time pulse sequences
optimized for proteins that allow selective refocusing of the carbonyl (e.g. hsqcctetgpsp,
hsqcctetgpsisp ). Run enough increments to see the carbon-carbon splitting in the standard
experiment. In both cases, use a refocusing nitrogen pulse to eliminate the effect of nitrogen
coupling in the indirect dimension (flag LABEL_CN).
C) 1
H-31
P correlation in DNA
Measure 1
H-31
P correlation spectrum of 3’, 4’, and 5’,5’’ protons with the phosphate backbone in the
DNA sample. Use the pulse sequence hp_rf. Refer to the proton and phosphorus 1D spectra
measured previously for the spectral widths and pulse lengths. As there is no signal in the first
increment in this type of experiment, check with d0 set to 50 ms before starting the measurement
(pulse sequence hp_rf_tst).