This project involves development of a new method, Multi-wavelength Anomalous Diffraction using Medium Angle X-ray solution scattering (MADMAX), for the precise measurement of interatomic distances within proteins and other macromolecules in aqueous solution. It will make possible the characterization of structural changes and intra-molecular movements that cannot be studied by existing techniques. Proteins are dynamic molecules whose activities contribute to all biological processes. Virtually all protein function requires intra- molecular movement, whether for the application of mechanical force, chemical transformation or molecular translocation. MADMAX will make possible accurate atomic-level measurement of these movements within proteins in solution. MADMAX should be generally applicable to macromolecules in solution and capable of elucidating protein action during a broad range of phenomena including allosteric interactions;protein-ligand interactions;channel gating;domain movements;and protein folding. It should be capable of measuring changes in the length of interatomic vectors with an accuracy of up to ~1 ?. It can be adapted for time- resolved studies - to at least millisecond resolution. The goals of this project are: (i) Demonstrate the accurate measurement of anomalous differences in solution scattering from well characterized proteins. (ii) Predict the anomalous differences from atomic coordinate sets. (iii) Determine the practical range of the method: How large a protein is this method applicable to? How many labels are required for detection? How much disorder in the label position can be tolerated? (iv) Demonstrate the use of MADMAX for precise measurement of structural changes due to ligand binding. (v) Demonstrate the use of MADMAX for the study of structural changes in membrane proteins. (vi) Demonstrate the use of time-resolved MADMAX for study of protein conformational changes. Taking full advantage of the capabilities of bacterial expression systems and chemical synthesis of proteins for the introduction of labels, the range of experiments that will be possible is almost limitless. Development of its full range of capabilities will establish MADMAX as an important new approach to the observation of protein structure and function in aqueous solution. This project will result in the development of a novel tool for the precise measurement of interatomic distances within proteins and the motions that they undergo during action. Development of advanced therapies depends on a deep understanding of these molecular actions because they form the basis of virtually all biological functions. Application of this approach to molecular systems involved in pathogenesis will contribute significantly to the development of strategies for the diagnosis and treatment of a broad range of human diseases.
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