Small-Angle Scattering Techniques for Analyzing Conformational Transitions in Hemocyanins

Abstract

The precise delivery of oxygen from respiratory surfaces to the tissues is mediated by cooperative and allosterically regulated carrier proteins, such as hemoglobin or hemocyanin. To establish cooperativity, these proteins must be able to adopt different conformations. These conformations are characterized by different ligand affinities, which have their basis in different structures as is the case for the deoxy and oxy states of human hemoglobin. To understand the cooperative interaction of these molecules at the molecular level, the structures of these conformations must be resolved and the transitions between them must be monitored. Because of the nature of sample preparation necessary for various methods, artificial forces may have an impact on the structure. X-ray structures are obtained from crystals. Electron microscopy delivers pictures from biomolecules either fixed on a surface and/or exposed to strong surface forces due to staining or ice buildup. These limitations also hold for newer techniques, such as atomic force microscopy (AFM). Two methods are available to study biomolecules in solution under in vivo conditions: high-resolution nuclear magnetic resonance (NMR) and small-angle scattering (SAS). NMR delivers structures with atomic resolution from rather small bio-molecules with a molecular mass less than 50 kDa. This is not the case for SAS. Here the resolution of the biomolecules obtained by this one- dimensional method is low but sufficient for large biomolecules. There are two methods of SAS— small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), each of which has its own advantages. SAXS experiments allow better angular resolution, whereas SANS enables the investigation of larger biomolecules. This chapter presents a methodical approach with SAS to obtain information about the large cooperative hemocyanins and their structural transitions on binding the ligand oxygen or allosteric effectors.