Tracing transport of protein aggregates in microgravity versus unit gravity crystallization.

Microgravity conditions have been used to improve protein crystallization from the early 1980s using advanced crystallization apparatuses and methods. Early microgravity crystallization experiments confirmed that minimal convection and a sedimentation-free environment is beneficial for growth of crystals with higher internal order and in some cases, larger volume. It was however realized that crystal growth in microgravity requires additional time due to slower growth rates. The progress in space research via the International Space Station (ISS) provides a laboratory-like environment to perform convection-free crystallization experiments for an extended time. To obtain detailed insights in macromolecular transport phenomena under microgravity and the assumed reduction of unfavorable impurity incorporation in growing crystals, microgravity and unit gravity control experiments for three different proteins were designed. To determine the quantity of impurity incorporated into crystals, fluorescence-tagged aggregates of the proteins (acting as impurities) were prepared. The recorded fluorescence intensities of the respective crystals reveal reduction in the incorporation of aggregates under microgravity for different aggregate quantities. The experiments and data obtained, provide insights about macromolecular transport in relation to molecular weight of the target proteins, as well as information about associated diffusion behavior and crystal lattice formation. Results suggest one explanation why microgravity-grown protein crystals often exhibit higher quality. Furthermore, results from these experiments can be used to predict which proteins may benefit more from microgravity crystallization.

Structure of nicotinic acid mononucleotide adenylyltransferase from Bacillus anthracis.

Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is the penultimate enzyme in the biosynthesis of NAD(+) and catalyzes the adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable candidate for antibacterial drug development; as such, Bacillus anthracis NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for the purpose of inhibitor discovery and crystallography. The crystal structure of BA NaMNAT was determined by molecular replacement, revealing two dimers per asymmetric unit, and was refined to an R factor and R(free) of 0.228 and 0.263, respectively, at 2.3 A resolution. The structure is very similar to that of B. subtilis NaMNAT (BS NaMNAT), which is also a dimer, and another independently solved structure of BA NaMNAT recently released from the PDB along with two ligated forms. Comparison of these and other less related bacterial NaMNAT structures support the presence of considerable conformational heterogeneity and flexibility in three loops surrounding the substrate-binding area.

Preliminary crystallographic studies of yeast mitochondrial peripheral membrane protein Tim44p.

Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of the TIM23 translocon. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p was crystallized. The crystals diffract to 3.2 A using a synchrotron X-ray source and belong to space group P6(3)22, with unit-cell parameters a = 124.25, c = 77.83 A. There is one Tim44p molecule in one asymmetric unit, which corresponds to a solvent content of approximately 43%. Structure determination by MAD methods is under way.