Carboxylic acids in crystallization of macromolecules: learning from successful crystallization experiments.

The production of macromolecular crystals suitable for structural analysis is one of the most important and limiting steps in the structure determination process. Often, preliminary crystallization trials are performed using hundreds of empirically selected conditions. Carboxylic acids and/or their salts are one of the most popular components of these empirically derived crystallization conditions. Our findings indicate that almost 40 % of entries deposited to the Protein Data Bank (PDB) reporting crystallization conditions contain at least one carboxylic acid. In order to analyze the role of carboxylic acids in macromolecular crystallization, a large-scale analysis of the successful crystallization experiments reported to the PDB was performed. The PDB is currently the largest source of crystallization data, however it is not easily searchable. These complications are due to a combination of a free text format, which is used to capture information on the crystallization experiments, and the inconsistent naming of chemicals used in crystallization experiments. Despite these difficulties, our approach allows for the extraction of over 47,000 crystallization conditions from the PDB. Initially, the selected conditions were investigated to determine which carboxylic acids or their salts are most often present in crystallization solutions. From this group, selected sets of crystallization conditions were analyzed in detail, assessing parameters such as concentration, pH, and precipitant used. Our findings will lead to the design of new crystallization screens focused around carboxylic acids.

Comparative structural and mechanistic studies of 4-hydroxy-tetrahydrodipicolinate reductases from Mycobacterium tuberculosis and Vibrio vulnificus.

BACKGROUND: The products of the lysine biosynthesis pathway, meso-diaminopimelate and lysine, are essential for bacterial survival. This paper focuses on the structural and mechanistic characterization of 4-hydroxy-tetrahydrodipicolinate reductase (DapB), which is one of the enzymes from the lysine biosynthesis pathway. DapB catalyzes the conversion of (2S, 4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate (HTPA) to 2,3,4,5-tetrahydrodipicolinate in an NADH/NADPH dependent reaction. Genes coding for DapBs were identified as essential for many pathogenic bacteria, and therefore DapB is an interesting new target for the development of antibiotics. METHODS: We have combined experimental and computational approaches to provide novel insights into mechanism of the DapB catalyzed reaction. RESULTS: Structures of DapBs originating from Mycobacterium tuberculosis and Vibrio vulnificus in complexes with NAD+, NADP+, as well as with inhibitors, were determined and described. The structures determined by us, as well as currently available structures of DapBs from other bacterial species, were compared and used to elucidate a mechanism of reaction catalyzed by this group of enzymes. Several different computational methods were used to provide a detailed description of a plausible reaction mechanism. CONCLUSIONS: This is the first report presenting the detailed mechanism of reaction catalyzed by DapB. GENERAL SIGNIFICANCE: Structural data in combination with information on the reaction mechanism provide a background for development of DapB inhibitors, including transition-state analogues.

Impact of an N-terminal Polyhistidine Tag on Protein Thermal Stability.

For years, the use of polyhistidine tags (His-tags) has been a staple in the isolation of recombinant proteins in immobilized metal affinity chromatography experiments. Their usage has been widely beneficial in increasing protein purity from crude cell lysates. For some recombinant proteins, a consequence of His-tag addition is that it can affect protein function and stability. Functional proteins are essential in the elucidation of their biological, kinetic, structural, and thermodynamic properties. In this study, we determine the effect of N-terminal His-tags on the thermal stability of select proteins using differential scanning fluorimetry and identify that the removal of the His-tag can have both beneficial and deleterious effects on their stability.