Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain.

Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent ß-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

NMR-assignments of a cytosolic domain of the C-terminus of polycystin-2.

Mutations in the PKD2 gene lead to the development of polycystic kidney disease (PKD). The PKD2 gene codes for polycystin-2, a cation channel with unknown function. The cytoplasmic, C-terminal domain interacts with a large number of proteins including mDia1, alpha-actinin, PIGEA-14, troponin, and tropomyosin. The C-terminal fragment polycystin-2 (680-796) consisting of 117 amino acids contains a putative calcium binding EF-hand. It was produced in Escherichia coli and enriched uniformly with (13)C and (15)N. The backbone and side chain resonances were assigned by multidimensional NMR methods, the obtained chemical shifts are typical for a partially folded protein. The chemical shifts obtained are in line with the existence of two paired helix-loop-helix (HLH) motifs.

Combined chemical shift changes and amino acid specific chemical shift mapping of protein-protein interactions.

Protein-protein interactions are often studied by chemical shift mapping using solution NMR spectroscopy. When heteronuclear data are available the interaction interface is usually predicted by combining the chemical shift changes of different nuclei to a single quantity, the combined chemical shift perturbation Deltadelta comb In this paper different procedures (published and non-published) to calculate Deltadelta comb are examined that include a variety of different functional forms and weighting factors for each nucleus. The predictive power of all shift mapping methods depends on the magnitude of the overlap of the chemical shift distributions of interacting and non-interacting residues and the cut-off criterion used. In general, the quality of the prediction on the basis of chemical shift changes alone is rather unsatisfactory but the combination of chemical shift changes on the basis of the Hamming or the Euclidian distance can improve the result. The corrected standard deviation to zero of the combined chemical shift changes can provide a reasonable cut-off criterion. As we show combined chemical shifts can also be applied for a more reliable quantitative evaluation of titration data.