Hello all,
I'd like to know anyone had the experience on carrying out 4D
13C,15N-separated HMQC-NOESY-HSQC experiment and/or the experience on
solving the structure of dimer. Here is my puzzle:
I'm trying to determine the interfacial NOE of a dimer ( 97 a.a.
monomer, m.w. ~11,000). I have prepared an 8mg/500uL equal molar ratio
15N/13C asymmetric labelled sample (4mg 15N labelled, 4mg 13C labelled,
mixed and denatured by 6M GdmHCl, incubated at 4 degree overnight, then
dialysized to renature the protein), then I carry out the 1D version of
a 4D 13C,15N HMQC-NOESY-HSQC (Muhandiram, Xu, Kay: J. Biomol. NMR 3,
463-70 (1993)), i.e. acquire the first fid of a 4D experiment.
To my surprise, the s/n ratio is very low even I accumulate 2048
transients. Actually the s/n is 2 after 2k scans. For a comparison, I
found the s/n of a 15N,13C double labelled sample (8mg/500uL too,
identical acquisition parameters) to be 31 after 32 scans for a 1D
version HMQC-NOESY-HSQC.
I have suspected the protein to be a monomer, but size exclusion
chromatography data show the protein to be larger than a 150 a.a.
protein. And I also found Tracy M. Handel had used a ns=2048 for 2D
version of the 4D experiment to identify interfacial NOE for MCP-1
protein, (yet ns=64 for a 3D version)
Any hint is greatly appreciated.
Yu-Feng
--
Yu-Feng Tong||童宇峰
NMR Group, Dept. Enzymology
Inst. Biophysics, Academia Sinica
15 Datun Road, Beijing, P.R.China, 100101
_______________________________________________________________________________
Life is an everlasting game of Weiqi(igo, baduk).
Received on Tue Mar 05 2002 - 08:59:41 MST