Table 1.

Determination of the of mass, size and composition of isolated macromolecules and macromolecular complexes in dilute solution

The molar mass of the chaperone protein trigger factor was determined by SEC-MALS in the absence and presence of a fragment of unfolded alkaline phosphatase. [58]. It was found that isolated trigger factor is dimeric in solution, but dissociates to monomer in order to bind unfolded protein.

SEC-MALS was used to establish that Ctf4, which acts as a linker protein in the eukaryotic replisome, is a trimer in solution, in agreement with the X-ray crystal structure. [59]

The molar mass of a solubilized membrane protein, nhTMEM16,encapsulated within a micelle of surfactant, was determined by size exclusion chromatography in conjunction with MALS, UV, and RI detectors. [60] Global analysis of signals from the three detectors indicated that the solubilized protein was present as a dimer, in agreement with the X-ray crystal structure.

SEC-MALS was used to measure the molar masses of a ubiquitin trimer (21.4 kDa), a caspase activation domain (23.2 kDa) and the tight complex formed by mixing the two (112.5 kDa) [61]. The results are in quantitative agreement with a model complex consisting of a tetramer of caspase activation domains binding a single ubiquitin trimer.

The molar mass of a complex of a myosin fragment (Myo4p) and an adaptor molecule (She3p) was measured via SEC-MALS and found to be equal to that of the hetero- trimer Myo4p + 2 She3p. [62] Additional SEC-MALS experiments on mixtures of Myo4, She3p, a second adaptor protein (She2p) and/or ribonucleotide signaling sequences referred to as zipcodes revealed the presence of higher-order complexes. However, the molar masses of the complexes so detected were strongly dependent upon solute concentration, indicative of reversible association, rendering the interpretation of these SEC-MALS chromatograms ambiguous, and qualitative at best. Observations such as these should be followed up by CG-MALS studies (Sections 2.3 and 3.4) in order to obtain unambiguous information about the strength and stoichiometry of equilibrium associations.

SEC-MALS was used to determine the molar masses of complexes of the Cμ4 domain of immunoglobulin IgM [63]. When a particular cysteine residue is deleted, this domain forms a noncovalent dimer in dilute solution. In the native domain covalently linked dimers are found that exist in slow equilibrium with hexamers of dimers. Analogous complexes of Cμ4 domains are found within the intact immunoglobulin, indicating that the overall quaternary structure of IgG derives from oligomerization of this domain.