A systematic analysis of backbone amide assignments achieved via combinatorial selective labelling of amino acids.

With the advent of high-yield cell-free expressions systems, many researchers are exploiting selective isotope labelling of amino acids to increase the efficiency and accuracy of the NMR assignment process. We developed recently a combinatorial selective labelling (CSL) method capable of yielding large numbers of residue-type and sequence-specific backbone amide assignments, which involves comparing cross-peak intensities in 1H- 15N HSQC and 2D 1H- 15N HNCO spectra collected for five samples containing different combinations of 13C- and 15N-labelled amino acids [Parker MJ, Aulton-Jones M, Hounslow A, Craven C J (2004) J Am Chem Soc 126:5020-5021]. In this paper we develop a robust method for establishing the reliability of these assignments. We have performed a detailed statistical analysis of the CSL data collected for a model system (the B1 domain of protein G from Streptococcus), developing a scoring method which allows the confidence in assignments to be assessed, and which enables the effects of overlap on assignment fidelity to be predicted. To further test the scoring method and also to assess the performance of CSL in relation to sample quality, we have applied the method to the CSL data collected for GFP in our previous study.

Cystatin forms a tetramer through structural rearrangement of domain-swapped dimers prior to amyloidogenesis.

The cystatins were the first amyloidogenic proteins to be shown to oligomerize through a 3D domain swapping mechanism. Here we show that, under conditions leading to the formation of amyloid deposits, the domain-swapped dimer of chicken cystatin further oligomerizes to a tetramer, prior to fibrillization. The tetramer has a very similar circular dichroism and fluorescence signature to the folded monomer and dimer structures, but exhibits some loss of dispersion in the 1H-NMR spectrum. 8-Anilino-1-naphthalene sulfonate fluorescence enhancement indicates an increase in the degree of disorder. While the dimerization reaction is bimolecular and most likely limited by the availability of a predominantly unfolded form of the monomer, the tetramerization reaction is first-order. The tetramer is formed slowly (t(1/2)=six days at 85 degrees C), dimeric cystatin is the precursor to tetramer formation, and thus the rate is limited by structural rearrangement within the dimer. Some higher-order oligomerization events parallel tetramer formation while others follow from the tetrameric form. Thus, the tetramer is a transient intermediate within the pathway of large-scale oligomerization.