Abstract

Periplasmic Nitrate Reductase (NapA/NapB) belongs to a class of proteins that are exported in a folded state by the twin arginine translocon (Tat) machinery. For a long time it was known that NapD is essential for the proper formation of the NapA/NapB enzyme. However, the mechanism by which this is accomplished is still unknown. The focus of this study is to understand the interaction between NapA and its maturation chaperone, NapD. For the first time we will show that the N-terminal consensus leader sequence of NapA that is involved in Tat export is also the binding site for the chaperone. To characterize the mechanism co-purification experiments, biophysical techniques and Nuclear Magnetic Resonance (NMR) were used. Co-purification experiments indicated that the N-terminal fragments; NapA114, NapA35 and NapA27 were able to bind NapD. Circular Dichroism (CD) analysis of NapD in complex with NapA35 illustrated a small increase in secondary structure. NMR confirmed that the small changes in CD spectra were due to conformational changes within NapD and the formation of an alpha-helix for NapA35. Chemical shift changes from NMR demonstrated that the entire leader peptide participates in binding with NapD. The residues that underwent the greatest changes in chemical shift are Arg6, Phe8, Met9, Lys10, Val14, Ala16-21, Gly22 and Leu23. NMR also showed that the NapA35 peptide is mobile when unbound. However when NapA35 is complexed with NapD, drastic changes in mobility occurred. For the first time the structure of the Tat leader peptide, NapA35 is determined when in complex with the chaperone NapD