Protocol

Protein Interactions Captured by Chemical Cross-linking: Two-Step Cross-linking with ANB•NOS

This protocol was adapted from "Protein Interactions Captured by Chemical Cross-linking," Chapter 7, in Protein-Protein Interactions (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

The two-step method of cross-linking with the photoreactive cross-linker ANB•NOS (7.7 Å) provides an additional layer of control over the one-step method with traditional bifunctional reagents. In this method, the initial modification and purification of one or more of the protein reactants can be carried out prior to cross-linking, thus minimizing both monoderivatization and nonspecific cross-linking. This method is also useful for detecting conformational changes in stable protein complexes. The complex can be labeled under solution conditions that favor, for example, an inactive conformer; it can then be purified and exchanged into a solution that favors an active conformation. In this case, any differences in cross-linking observed under the two conditions will reflect structural changes that occur at each site of modification on the complex, rather than conformation-dependent cross-linking at different sites (which is a more likely scenario when a complex is modified with the same cross-linking reagent under different reaction conditions).