th  - 1988 - 51st ENC Conference

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SEQUENTIAL ASSIGNMENT OF AMIDE PROTONSIN o~-HELICES IN LARGE PROTEINS
Steven W. Sparks +*, Ad Bax ++, and Dennis A. Torchla +
NIDR +, NIDDK++,National Institutes of Health, Bethesda, MD 20892
We describe an approach that yields sequential assignments of proton signals in u-helices in
proteins that are too large to apply the standard assignment strategy. Deuteration of
non-exchangeable protons is used to enhance dNN connectivities in the protein NOESY spectrum,
thereby revealing long sequences of dNN connectivities that are characteristic of (x-helices. Double
labeling with 13C/15N is used to edit and assign signals in proton detected heteronuclear shift
correlation (HMQC) spectra of the protein. The sequential assignments are obtained by comparing
i
the amide proton chemical shifts in the NOESY and HMQC spectra. We show that this meihod
provides assignments for all amide protons in the three (x-helical domains of staphylococcal
nuclease complexed with pdTp and Ca 2+, MW = 18 kDa. The fact that the assignments were obtained
at a low protein concentration (1.5 raM), and at physiological temperature (36.5 °) and pH (7.7),
indicates that this approach can be applied to a wide range of proteins. The HMC)C spectra also
provide assignments of protons outside of the (x-helices. We show that these assignments can be
used as starting points for sequential assignments of other structural domains in the protein.
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MASS TRANSFER PROCESSES STUDIED BY NMR IMAGING.
L.D.HALL AND A.G.WEBB*
Laboratory for Medicinal Chemistry,
Level 4, Radiotherapeutic Centre
Addenbrookes Hospital, Hills Road,
Cambridge. CB2 2QQ. England.
Diffusional processes play an important role in many chemical and
biological systems. Examples include the rate determination of chemical
reactions, and the mass transport properties of biological membranes. NMR
imaging can be used to follow these processes by exploiting the difference in
relaxation properties between the species of interest and the medium through
which it is diffusing. Quantitative information concerning diffusion
coefficients can then be calculated.
We will present examples of the diffusion of common solvents through
industrially important polymers such as polymethylmethacrylate. In addition we
have studied the temporal and spatial localisation of the aerially catalysed
reduction of hydroquinone to produce a semiquinone anionic free radical. This
series of radical anions play an important role in various biological pathways.
All experiments were carried out on an Oxford 2T wide-bore ( 31 cms. )
magnet. The maximum gradient strength of lOmT/m produced a slice thickness of
3.5mm. An inversion recovery spin echo refocussed imaging sequence was used to
produce T 1 weighted images.
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