The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix.

A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenase, named after their metal requirements, containing either a MoFe-, VFe- or FeFe-cofactor, that converts N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase proteins within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.

Electrospray ionization mass spectrometry of oligonucleotide complexes with drugs, metals, and proteins.

I. Introduction 61 II. Binding of Small Molecules to DNA 62 A. Covalent Binding 62 B. Reversible (Noncovalent) DNA-Binding Agents 65 III. DNA-Metal Ion Complexes 67 A. Platinum Complexes 70 B. Other Metal Ions 73 IV. DNA-Protein Complexes 74 A. Introduction 74 B. ESI-MS of DNA-Protein Complexes 76 C. ESI-MS Analysis of Proteolytic Products of DNA-Protein Complexes 79 D. ESI-MS of Ternary DNA-Protein-Ligand Complexes 80 V. Conclusions 80 Abbreviations 81 References 81 --Interactions of DNA with drugs, metal ions, and proteins are important in a wide variety of biological processes. With the advent of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), mass spectrometry (MS) is now a well-established tool for the characterization of the primary structures of biopolymers. The gentle nature of the ESI process, however, means that ESI-MS is also finding application for the study of noncovalent and other fragile biomolecular complexes. We outline here the progress, to date, in the use of ESI-MS for the study of noncovalent drug-DNA and protein-DNA complexes together with strategies that can be employed to examine the binding of small molecules and metal complexes to DNA. In the case of covalent complexes with DNA, sequence information can be derived from ESI-MS used in conjunction with tandem mass spectrometry (MS/MS) and/or enzymatic digestion. MS/MS can also be used to probe the relative binding affinities of drugs that bind to DNA via noncovalent interactions. Overall, the work in this area, to date has demonstrated that ESI-MS and MS/MS will prove to be valuable complements to other structural methods, offering advantages in terms of speed, specificity, and sensitivity. (c) 2001 John Wiley & Sons, Inc.