Application and development of multi-dimensional NMR spectroscopic techniques to study protein structure in solution.

Abstract

The solution structure of the periplasmic cyclophilin type cis-trans peptidyl-prolyl isomerase from Escherichia coli (167 residues, Mr = 18,244 daltons) has been determined using multi-dimensional heteronuclear NMR spectroscopy and distance geometry calculations. The structure determinationa is based on a total 968 NMR derived restraints (67 $\phi$ torsion angle restraints and 901 distance restraints). A total of seven distance geometry structures were calculated, and the average root mean square distribution about the mean backbone coordinate positions is 1.67 A for the backbone atoms (residues 5-164) of the ensemble. The three dimensional structure of the cis trans peptidyl-prolyl isomerase consists of an eight stranded anti-parallel $\beta$-sheet barrel capped by two alpha helices. The putative binding site for peptide and protein substrates shares similar topology to human T-cell cyclophilin. In contrast to human cyclophilin, two surface loops in the active site of the E. coli protein appear to exhibit large conformational fluctuations. Furthermore, a histidine residue essential for isomerase activity in human (H126) is replaced by a tyrosine residue (Y122) in E. coli cyclophilin. Proton and nitrogen-15 sequence specific nuclear magnetic resonance assignments have been determined for recombinant oxidized flavodoxin from Anacystis nidulans (169 residues, Mr = 19,048). In solution, flavodoxin consists of a five stranded parallel $\beta$-sheet involving residues 3-9, 31-37, 49-56, 81-89, 114-117, and 141-144. Medium range NOEs indicate that residues 12-21, 40-46, 103-114, 145-148, and 152-169 form helices in solution. The FMN binding site has been investigated using polypeptide-FMN NOEs. Three new multi-dimensional heteronuclear pulse schemes are presented. The correlation of adjacent residues based on the conformation independent derivation of $\sp1$H$\sp\alpha$ chemical shifts is accomplished with the HN(CA)HA and HN(COCA)HA experiments. Another new triple resonance experiment, the HN(CA)CO, correlates intraresidue amide proton, amide nitrogen and carbonyl nuclei and is complementary to the HNCO experiment. The utility of these experiments was demonstrated on a uniformly enriched sample of T4 lysozyme.