Structure of a switchable subtilisin complexed with a substrate and with the activator azide.

An engineered variant of the protease subtilisin from Bacillus amyloliquefaciens, in which the D32A mutation renders the enzyme's activity dependent on the presence of certain small anions such as fluoride or azide, has been produced. This modified enzyme has applications as an azide or fluoride-triggered expression-purification tool. We report activity measurements showing that the enzyme is activated more than 3000-fold by azide and describe the 1.8 A resolution structure of an inactive form (by replacing the catalytic nucleophile Ser 221 with alanine) of the protease, in complex with azide and with a substrate that spans the active site. Both enzyme and substrate have been engineered to increase their stability and the affinity of their interaction. The substrate is based on a stabilized subtilisin prodomain, extended across the active site by the addition of four residues at its C-terminus. In the crystal structure, the substrate is well-ordered across the active site, and the azide anion is observed bound adjacent to Ala 32. The structures of the substrate complex in three different crystals (anion-free, fluoride-soaked, and azide-soaked) are compared. These structures provide extensive information for understanding subtilisin's substrate binding and catalytic mechanism, and for the development of biotechnology tools based on anion-activated proteolysis. The mechanism of anion-dependent proteolysis appears to be a slight modification of the accepted charge-relay mechanism for serine proteases.

A Quality Improvement Approach to Increase Exercise Assessment in Survivors of Childhood Leukemia.

INTRODUCTION: Survivors of childhood cancer are at increased risk of treatment-related cardiovascular disease, the severity of which is impacted by the level of regular exercise. Exercise assessments (EAs) are not a routine component of follow-up care. METHODS: We incorporated a quantitative EA tool into the clinic triage during follow-up visits for survivors of acute lymphoblastic leukemia. The nursing staff was surveyed on the use of the EA tool to gauge understanding and level of comfort with addressing patient questions. RESULTS: Over 27 months, the percentage of off-therapy acute lymphoblastic leukemia patients with documented EA increased from 0% to 80%. We noted degradation in EA completions in the last 6 months of the project, which we attributed to project nursing staff transition and failure to maintain education. Interventions that improved the percentage of completed EA included the incorporation the assessment tool into the electronic medical record and weekly reminders of scheduled eligible patients. A nurse incentive plan did not impact project success. Survey results revealed that the nursing staff were comfortable with the EA and did not view the new process as hurting patient flow. CONCLUSION: Implementation of an EA tool into routine clinic follow-up was successful. We met the project goal of assessing greater than 50% of the follow-up patients. This work will serve as the foundation for the next phase of the project, which will be to provide education on the importance of exercise and earlier intervention when a sedentary lifestyle is identified.

Stray light correction in the optical spectroscopy of crystals.

It has long been known in spectroscopy that light not passing through a sample, but reaching the detector (i.e., stray light), results in a distortion of the spectrum known as absorption flattening. In spectroscopy with crystals, one must either include such stray light or take steps to exclude it. In the former case, the derived spectra are not accurate. In the latter case, a significant amount of the crystal must be masked off and excluded. In this paper, we describe a method that allows use of the entire crystal by correcting the distorted spectrum.