So how do you know you have a macromolecular complex?

Abstract

Protein in crystal form is at an extremely high concentration and yet retains the complex secondary structure that defines an active protein. The protein crystal itself is made up of a repeating lattice of protein-protein and protein-solvent interactions. The problem that confronts any crystallographer is to identify those interactions that represent physiological interactions and those that do not. This review explores the tools that are available to provide such information using the original crystal liquor as a sample. The review is aimed at postgraduate and postdoctoral researchers who may well be coming up against this problem for the first time. Techniques are discussed that will provide information on the stoichiometry of complexes as well as low-resolution information on complex structure. Together, these data will help to identify the physiological complex.

Figure 1

The process of crystallization may select nonphysiological protein associations. (a) The physiological state of the protein is a dimer and the dimer can be crystallized to provide a structure. (b) The physiological state of the protein is a dimer. The dimer cannot pack into a lattice to produce a crystal, but the monomer alone can. Therefore, the crystal structure contains the nonphysiological state. (c) and (d) demonstrate an analogous case where the physiological state is a monomer. For clarity, the physiological oligomerization state is circled.

Figure 2

A comparison of data from SEC (a) and AUC (b) on the same protein. SEC provides a weight that is close to that expected for a dimer, whereas AUC shows a peak for the weight of a tetramer. It is likely that the result using SEC indicates that the protein (which is membrane-associated) interacts with the column matrix, leading to retardation and an erroneously low estimation of weight.

Figure 3

The use of FRET to determine the correct dimer structure for α₁-antitrypsin (Sivasothy et al., 2000 ▶). (a) Models showing three possible dimer structures: I, II and III. The residues upon which fluorophores are attached are shown in dimer I. (b) The fluorescence of a donor fluorophore in the presence of an acceptor fluorophore is measured under conditions that promote and disrupt polymerization. FRET results in a decrease in the fluorescence from the donor fluorophore. A range of FRET signals are measured for proteins with fluorophores at different positions on the surface of α₁-antitrypsin. These are then used to model the structure of the dimer. The correct dimer is dimer III.