Calorimetry Work Station

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Eight BBRI scientists working in fields in which protein interactions are an important component, propose to purchase a Calorimetry Work Station consisting of different scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) instrumentation. The work station will enable them to determine the energetic contributions within and between biological macromolecules which is key to understanding the relationship between structure and function. The current experimental approaches of the investigators involve a variety of techniques including X-ray crystallography, fluorescence, ultracentrifugation, CD and other spectroscopic methods as well as immunological and other measurements of biological activity. Calorimetric methods for measuring the details of protein stability and protein interactions would provide more quantitative, and because of the thermodynamic parameters obtainable, a more basic description of the interactions involved in these studies. Thus, interactions that determine structure will be related to function. The Calorimetry Work Station in BBRI is needed to complement other structural and functional studies with muscle proteins, chaperones and immunologicals as detailed in the individual research plans and generally will be used to: (i) characterize stability of proteins and their modifications. This will help in the design of suitable mutants for functional and crystallographic studies. (ii) measure thermodynamic stability of protein domains. DSC is more useful than CD because the domains need not have much secondary structure, and multiple transitions which are present in these systems are easier to analyze. (iii) measure binding constants and characterize the thermodynamics of binding equilibria. ITC avoids the unavoidable perturbation of fluorescence labels and is more quantitatively than ELISA assays of binding.