Characterization of metalloproteins by high-throughput X-ray absorption spectroscopy.

High-throughput X-ray absorption spectroscopy was used to measure transition metal content based on quantitative detection of X-ray fluorescence signals for 3879 purified proteins from several hundred different protein families generated by the New York SGX Research Center for Structural Genomics. Approximately 9% of the proteins analyzed showed the presence of transition metal atoms (Zn, Cu, Ni, Co, Fe, or Mn) in stoichiometric amounts. The method is highly automated and highly reliable based on comparison of the results to crystal structure data derived from the same protein set.

Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex.

Nuclear pore complexes (NPCs), responsible for the nucleo-cytoplasmic exchange of proteins and nucleic acids, are dynamic macromolecular assemblies forming an eight-fold symmetric co-axial ring structure. Yeast () NPCs are made up of at least 456 polypeptide chains of ~30 distinct sequences. Many of these components (nucleoporins, Nups) share similar structural motifs and form stable subcomplexes.

Structure of a putative BenF-like porin from Pseudomonas fluorescens Pf-5 at 2.6 A resolution.

The X-ray structure of a putative BenF-like (gene name: PFL1329) protein from (PflBenF) has been determined at 2.6Å resolution. X-ray crystallography revealed a canonical 18-stranded β-barrel fold that forms a central pore with a diameter of ∼4.

Mass spectrometry guided in situ proteolysis to obtain crystals for X-ray structure determination.

A strategy for increasing the efficiency of protein crystallization/structure determination with mass spectrometry has been developed. This approach combines insights from limited proteolysis/mass spectrometry and crystallization via in situ proteolysis. The procedure seeks to identify protease-resistant polypeptide chain segments from purified proteins on the time-scale of crystal formation, and subsequently crystallizing the target protein in the presence of the optimal protease at the right relative concentration.