th  - 1988 - 51st ENC Conference

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2D NMR STUDIES AT 600 MHZ OF A PROTEIN-DNA COMPLEX USING IMPROVED TECHNIQUES
FOR WATER SUPPRESSION AND HETERONUCLEAR CORRELATION SPECTROSCOPY
C. OTTINC*, W. LEUPIN, A. EUCSTER, AND K. WOTHRICH, INSTITUT FOR
MOLEKULARBIOLOCIE UND BIOPHYSIK, ETH, CH-8093 ZORICH
The N-terminal DNA-binding domain 1-76 of the P22 c2 repressor was investigated
in a I:I complex with a 16-base pair DNA duplex related to the ORI binding
site. Starting from the sequence-specific resonance assignments previously
established for the isolated protein and the DNA duplex, resonance assignments
could be made for the complex using conventional 2D NMR techniques. For NOESY
in H20 an improved scheme was developed which suppresses the water resonance
without presaturation at the end of the mixing time, and provides uniform
excitation in both dimensions, except for a region of approximately +/-1.5 ppm
in ~2 centered about the water line. To obtain simplified IH-NMR spectra of the
complex, a protein preparation with 15N enriched lysine and arginine residues
was prepared. The resonances of the protons directly bound to 15N were then
selectively observed using 15N(~ 2) half-filtered NOESY. IH- detected 15 N
correlation spectroscopy was performed using the pulse sequence by Bodenhausen
and Ruben (Chem. Phys. Lett. 69, 185 (1980)). To record [IH,15N]-COSY spectra
the performance of the experiment was improved by insertion of short spin lock
pulses which purge all undesired signals originating from protons not directly
bound to 15N. The purge pulses enabled us to use this experiment also for
[IH,13C]-COSY at natural abundance with a 5mM solution of repressor 1-76, and
to extend the pulse sequence with a TOCSY-type mixing period to obtain relayed
correlations.
~ ALTERNATIVE METHODS FOR COLLECTION OF 2D-NMR SPECTRA: Peter
40 I Rinaldi* and Dan Iverson+, Department of Chemistry, The
University of Akron, Akron, OH 44325* and Varian Instruments, 611 Hansen Way,
Palo Alto, CA 94303+.
The standard method for obtaining 2D-NMR spectra involves collection of
all NT transients at a single evolution time before incrementing tl, where NT
is an integral multiple of cycles such as CYCLOPS to systematically reduce ar-
tifacts, and n is the number of t I increments.
(90x-O-90x-AT)NT, (90x-0-90y-AT)NT, (90x-T-9Ox-AT)NT,
(90x-T-90y-AT)NT, (90x-2T-9Ox-AT)NT, (90x-2T-90y-AT)NT,
.... (90x-nT-90x-AT)NT, (90x-nT-9Oy-AT)N T
When magnetization is not permitted to fully decay (as is typically the
case) this method produces systematic t I artifacts such as the characteristic
"false" COSY crosspeaks that appear from sharp singlets. Alternative orders
for collection of 2D-NMR data have been examined using the COSY sequence.
Collection of a single transient for all values of t I and repeating the se-
quence NT times provides considerably better artifact suppression and at the
same time requires fewer steady state cycles if NT is small.
[(90x-0-90x-AT), (90x-T-9Ox-AT), (90x-2T-90x-AT),
.... (90x-nT-9Ox-AT), (90x-0-90y-AT),
(90x-T-9Oy-AT), (90x-2T-90y-AT) .... , (90x-nT-90y-AT)]NT
Sample data obtained from trans-stilbene and methy methacrylate are
shown to illustrate the level of artifact reduction.
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