Purification and characterization of a type III photolyase from Caulobacter crescentus

The photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases, and cryptochromes that regulate blue-light-dependent growth and development in plants, and light-dependent and light-independent circadian clock setting in animals. Phylogenetic analysis has revealed a new class of the family, named type III photolyase, which cosegregates with plant cryptochromes. Here we describe the isolation and characterization of a type III photolyase from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either both chromophores or only folate, we were able to determine the efficiency and rate of transfer of energy from MTHF to FAD.     

Nutritional status and energy metabolism of crayfish (Procambarus clarkii, Girard) muscle and hepatopancreas

1. Food deprivation resulted in significant decreases in muscle carbohydrate, lipid and water content and increased ATP, ADP, AMP and total adenylate levels over the 21-day experimental period. 2. In the hepatopancreas phosphoarginine was significantly higher on day 21 in the starved crayfish. 3. Muscle energy charges remained within optimal (unstressed) ranges, while hepatopancreatic energy charges of food-deprived crayfish fell into suboptimal (stressed) ranges, indicating the necessity of examining organs separately to accurately ascertain metabolic changes in response to stressors.     

Computational active site analysis of molecular pathways to improve functional classification of enzymes

This study describes a method to computationally assess the function of homologous enzymes through small molecule binding interaction energy. Three experimentally determined X-ray structures and four enzyme models from ornithine cyclo-deaminase, alanine dehydrogenase, and mu-crystallin were used in combination with nine small molecules to derive a function score (FS) for each enzyme-model combination. While energy values varied for a single molecule-enzyme combination due to differences in the active sites, we observe that the binding energies for the entire pathway were proportional for each set of small molecules investigated. This proportionality of energies for a reaction pathway appears to be dependent on the amino acids in the active site and their direct interactions with the small molecules, which allows a function score (FS) to be calculated to assess the specificity of each enzyme. Potential of mean force (PMF) calculations were used to obtain the energies, and the resulting FS values demonstrate that a measurement of function may be obtained using differences between these PMF values. Additionally, limitations of this method are discussed based on: (a) larger substrates with significant conformational flexibility; (b) low homology enzymes; and (c) open active sites. This method should be useful in accurately predicting specificity for single enzymes that have multiple steps in their reactions and in high throughput computational methods to accurately annotate uncharacterized proteins based on active site interaction analysis.     

Using the protein chip interface with quadrupole time‐of‐flight mass spectrometry to directly identify peaks in SELDI profiles – initial evaluation using low molecular weight serum peaks

Mass spectrometric profiling, particularly in the form of SELDI, has been used in many studies, particularly in attempts to generate diagnostic serum profiles. Several studies have generated promising results but one of the limitations is the inability to identify easily potential discriminatory peaks. This may enable specific assays to be developed and increased biological insight. We describe the first systematic technical evaluation of the ProteinChip interface coupled to a tandem mass spectrometer which allows direct sequencing of peptides <6000 Da, and describe the direct sequence identification of 21 peaks commonly observed in serum samples. Additionally we describe for the first time the use of on‐chip acetylation to assist in the validation of sequence identification.