Time-Resolved Small-Angle X-Ray Scattering for Monitoring the Biomolecular Assembly Process

The small-angle X-ray scattering (SAXS) is a useful technique to know the assembly state of biomolecules. We can obtain the molecular weight of the assembled complex from the forward scattering intensity, that is, we can know how many molecules are included in the complex. The scattering profile also provides the information about the shape of the complex. The development of a photon-counting PILATUS detector made it possible to observe two-dimensional scattering images at every few milliseconds. Using this detector and a stopped-flow apparatus, we followed the assembly reactions of Escherichia coliferritin A (EcFtnA) and its mutants by the time-resolved SAXS (TR-SAXS) method. EcFtnA forms a cage-like structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains native-like secondary and tertiary structures and is able to reassemble into a 24-mer when the pH is increased. The reassembly reaction was induced by pH jump and reassembly was followed by TR-SAXS. Time-dependent changes in the forward scattering intensity and in the radius of gyration suggested the existence of a significant population of intermediate oligomers during the assembly reaction. The initial reaction was a mixture of second- and third-order reactions (formation of tetramers and hexamers) from the protein-concentration dependence of the initial velocity. The time-dependent change in SAXS profile could be explained by a simple model in which only tetramers, hexamers, and dodecamers were considered as intermediate.