th  - 1987 - 51st ENC Conference

WK2
15N NMR OF MACROMOI_F.CULF-S: APPLICATIONS TO THE
b"I'UDY OF PROTEINS IN SOLUTION
M. Bogusky', P. Leighton, R. Schiksnis, A. Khoury, P. Lu and S. Opella.
Department of Chemistry
University of Pennsylvania
Philadelphia, Pennsylvania 19104
One- and two-dimensional heteronuclear NMR techniques
combined with 15N biosynthetic labelling have been used to study a variety
of globular and membrane bound proteins in solution. Five proteins
including the fd and Pfl phage coat proteins in micelles, cro repressor, lac
repressor and lac headpiece, covering the molecular weight range
6-155kD have been investigated using these techniques. Heteronuclear
correlation, both nitrogen and proton observe are used to resolve amide
nitrogens and protons as well as sidechains containing nitrogen.
Assignment of resonances to amino acid type are completed through the
use of single site 15N biosynthetic labelling. Sequence specific assignments
are accomplished with the use of 13C-15N double labelled samples.
Evaluation of the advantages and limitations of both heteronuclear
correlation techniques with regard to molecular weight are presented.
Applications of polarization transfer techniques are used to
increase sensitivity, assign resonances and monitor proton exchange for the
labelled proteins discussed. Protein backbone and sidechain dynamics are
characterized by the heteronuclear 15N-(IH} NOE. Qualitative
determination of protein mobility on the nanosecond timescale is readily
accomplished for moderately sized proteins using the heteronuclear NOE.
Proteins dynamics are also characterized with the use of "dynamic filters"
inherent in several heteronuclear multipulse experiments. The combination
of the heteronuclear techniques discussed with the conventional proton
NMR techniques extend the molecular weight range of protein systems
which become tractable by high resolution solution NMR.