| Abstrakt: |
Multi-angle laser light scattering (MALLS) is a valuable spectroscopic technique that can give the absolute mass of macromolecules, independent of molecular shape. When coupled with size exclusion chromatography (SEC), individual components may be separated into fractions before analyses by MALLS, thus yielding the absolute molecular mass of each species. This review covers the technical background for MALLS-SEC as well as several examples of its application from our own work. We have utilized MALLS-SEC to investigate the protein-protein and protein-DNA interactions of the VDJ recombinase. The recombination activating gene products (RAG1 and 2) form the VDJ recombinase and catalyze the initial DNA cleavage steps during V(D)J recombination, the mechanism primarily responsible for diversification of the immune response. Through MALLS-SEC analysis of the RAG proteins, we have determined the self-association properties of RAG1, investigated ionic interactions in oligomerization, and performed kinetic studies of oligomerization over time. In addition, we have analyzed the oligomerization of RAG1 in the presence of its target DNA, the recombination signal sequence. Finally, we provide an overview of the current applications of MALLS-SEC from recent literature, which illustrates the scope of MALLS-SEC as a powerful technique for determining the biophysical properties of macromolecules. |
