S. Grzesiek, H. Kuboniwa, N. Tjandra, A. Wang, R. Tschudin, L. Nicholson, D. Torchia and A. Bax
National Institutes of Health, Bethesda, MD 20892-0510, USA
Uniform isotopic enrichment of macromolecules with 13C and 15N makes it possible to record highly sensitive three- and four-dimensional NMR spectra in which resonance overlap is dramatically reduced. This technology has moved the upper molecular weight limit for protein structure determination into the 20 to 40 kDa range. At the same time, the isotope labelling has given access to a whole range of additional structural and dynamical parameters such as heteronuclear chemical shifts, J-couplings and heteronuclear relaxation times.
We show how these new techniques are applied to the study of the structure and dynamics of HIV-1 protease/inhibitor complexes and of calmodulin whose binding affinity for its target enzymes is modulated by calcium. A very detailed picture of the calcium-free state of this protein has been determined based on an unusally large number of parameters. This picture permits a detailed comparison with the calcium-bound state of the protein whose structure has been reported previously.
Spectral line broadening which ultimately limits the accessible molecular weight for high resolution NMR is caused primarily by dipolar coupling to protons. Additional isotopic enrichment with 2H narrows the line widths and reduces magnetic "spin diffusion" through the proton spin reservoir. This can be used to record high quality four-dimensional 15N-15N-edited NOE spectra of the residual amide protons even for proteins with large rotational correlation times, such as the HIV-1 protein Nef. The large number of uniquely determined distance constraints determined from those spectra allows a quick placement of Nef's secondary structure elements relative to each other in a 3-dimensional topology.