On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications.

Within the complex milieu of a cell, which comprises a large number of different biomolecules, interactions are critical for function. In this post-reductionist era of biochemical research, the 'holy grail' for studying biomolecular interactions is to be able to characterize them in native environments. While there are a limited number of in situ experimental techniques currently available, there is a continuing need to develop new methods for the analysis of biomolecular complexes that can cope with the additional complexities introduced by native-like solutions. We think approaches that use microfluidics allow researchers to access native-like environments for studying biological problems. This review begins with a brief overview of the importance of studying biomolecular interactions and currently available methods for doing so. Basic principles of diffusion and microfluidics are introduced and this is followed by a review of previous studies that have used microfluidics to measure molecular diffusion and a discussion of the advantages and challenges of this technique.

The First Purification of Functional Proteins from the Unculturable, Genome-Reduced, Bottlenecked α-Proteobacterium 'Candidatus Liberibacter solanacearum'.

'Candidatus Liberibacter solanacearum' is an unculturable α-proteobacterium that is the causal agent of zebra chip disease of potato-a major problem in potato-growing areas, because it affects growth and yield. Developing effective treatments for 'Ca. L. solanacearum' has been hampered by the difficulty in functionally characterizing the proteins of this organism, largely because they are not easily expressed and purified in standard expression systems. 'Ca. L. solanacearum' has a reduced genome and its proteins are predicted to be prone to instability and aggregation. Among intracellular-dwelling bacteria, chaperone proteins are conserved and overexpressed to buffer against problems in protein folding. We mimicked this approach for expressing and purifying 'Ca. L. solanacearum' proteins in Escherichia coli by coexpressing them with chaperones. Neither of the representative 'Ca. L. solanacearum' enzymes, dihydrodipicolinate synthase (key in lysine biosynthesis) and pyruvate kinase (involved in glycolysis), were overexpressed in standard E. coli expression plasmids or strains. However, soluble dihydrodipicolinate synthase was successfully coexpressed with GroEL/GroES, while soluble pyruvate kinase was successfully coexpressed with either GroEL/GroES, dnaK/dnaJ/grpE, or a trigger factor. Both enzymes, believed to be key proteins for the organism, were purified by a combination of affinity chromatography and size-exclusion chromatography. Additionally, both 'Ca. L. solanacearum' enzymes are active and have the canonical tetrameric oligomeric structure in solution, consistent with other bacterial orthologs. This is the first study to successfully isolate and functionally characterize proteins from 'Ca. L. solanacearum'. Thus, we provide a general strategy for characterizing its proteins, enabling new research and drug discovery programs to study and manage the pathogenicity of the organism.

A new lysine biosynthetic enzyme from a bacterial endosymbiont shaped by genetic drift and genome reduction.

The effect of population bottlenecks and genome reduction on enzyme function is poorly understood. Candidatus Liberibacter solanacearum is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid-a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the function of Ca. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyzes the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate that CLsoDHDPS is expressed in Ca. L. solanacearum and expression is increased ~2-fold in the insect host compared to in planta. CLsoDHDPS has decreased thermal stability and increased aggregation propensity, implying mutations have destabilized the enzyme but are compensated for through elevated chaperone expression and a stabilized oligomeric state. CLsoDHDPS uses a ternary-complex kinetic mechanism, which is to date unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure of CLsoDHDPS with both pyruvate and the substrate analogue succinic-semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study suggests the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria-host co-evolutionary associations. We propose that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts.