Toward the solution structure of large (>30 kDa) proteins and macromolecular complexes

Many of the NMR experiments fail to determine the solution conformations for molecular weights in excess of ∼30 kDa. This chapter describes strategies and their applications to the 37 kDa complex between E.coli trp repressor and trp operator, and discusses the limitations of this method and promising recent results suggesting that the strategies mentioned above will extend the usefulness of NMR to molecular weights in the range of 30–40 kDa. The chapter discusses two strategies that can be used to extend the usefulness of NMR to larger macromolecules and complexes; first, one can modify current sequences to minimize the length of the pulse sequence, and secondly, one can improve the sensitivity of pulse schemes by increasing the T₂ relaxation times of nuclei involved through random incorporation of deuterium into the protein. From preliminary NMR measurements, it appears that the level of deuterium incorporation at the Cα position is approximately that of the deuterium level in the growth media. The incorporation of deuterium within the amino acid side chains is currently under investigation. It is likely that a complete backbone assignment will be possible with complementary triple resonance experiments on this deuterated complex. This is a promising result indicating that the use of fractional deuteration may enable one to assign proteins and complexes as large as perhaps 30–40 kDa.