Table 4.

Characterization of specific associations in dilute solution

DLS was used to detect heteroassociation of a trypsin/chymotrypsin inhibitor, BTCI, and the 20S proteasome [87]. Unfortunately, no measurements of the dependence of DLS upon the composition of BTCI – proteasome mixtures was performed, so a composition gradient analysis is not possible.

CG-MALS was used to characterize the equilibrium association between streptavidin and an anti-streptavidin antibody [88] Due to the multivalent nature of both streptavidin and antibody, a variety of large streptavidin-antibody complexes may be formed. The dependence of weight-average molar mass upon the concentrations of streptavidin and antibody may be accounted for quantitatively with an analytically soluble model containing three association constants and the molar masses of the two macromolecular reactants.

A review of various methods, including CG-SLS and DLS, used to characterize the self-association of the bacterial division protein FtsZ under various conditions was published [89]. This review provides an excellent example of how light scattering can be used in conjunction with complementary techniques to provide a comprehensive, multidimensional picture of a thoroughly studied self-associating protein.

SEC-MALS was used to verify the formation of physical complexes of proteins involved in base excision DNA repair and to determine their stoichiometries [90]. DLS was used to estimate the sizes of the complexes.

CG-MALS was used to characterize the reversible monomer-dimer equilibrium of the N-terminal domain of the bicarbonate transporter protein NBCel-A [91]. A disease- related mutant of this domain was found to aggregate irreversibly under Zoidentical conditions.

CG-DLS and CG-MALS were used to measure the effect of variation in temperature, pH, buffer type, and Hofmeister anions upon the reversible self-association of a monoclonal antibody [92]. The self association scheme best describing the data (monomer – trimer – (trimer)2 – (trimer)3 - …) was found to be unaffected by changes in conditions, and the same as characterized previously [35], but equilibrium constants and corresponding relative stabilities of these states were affected. For example, increasing temperature was found to inhibit self-association and increasing salt concentration promoted self-association.

CG-MALS was used to characterize associations between a peptide derived from the calcium-activated potassium channel (SKp) and calmodulin (CaM) in the absence and presence of saturating Ca and at low and high protein concentrations [93]. Systematic variation of solution composition revealed that In the presence of 2 mM Ca, SKp and CaM were found to reversibly form complexes with SKp/CaM stoichiometries of 1:1, 2:1, and 1:2. In the absence of Ca the 1:2 complex was not observed.

DLS was employed together with differential scanning calorimetry to examine the relationship between temperature, self-association, thermal stability, and ligand binding of folate binding protein (FBP) [94]. The authors inferred changes in the state of association of FBP from changes in the apparent hydrodynamic radius and the distribution of hydrodynamic radii reported by the DLS instrumentation. Estimates of molar mass and states of association were presented without explanation of how the values were derived from DLS data.

SEC-MALS and CG-MALS were used to determine the states of association of signal transduction proteins Vie-A-His6, Vie-B, and VieS-C, and to detect and quantify reversible hetero-association between various mixtures of these proteins [95]. Each pair of proteins except VieA-His6/VieB was reported to combine to form complexes of distinct stoichiometry.

CG-DLS was used to quantitatively characterize the effects of N-trimethylamine oxide (TMAO) and guanidine hydrochloride (GuHCl) on the reversible dissociation of the α2β2 tetramer of cyanmethemoglobin into αβ dimers [14]. The dependence of the z- average diffusion coefficient upon the concentrations of TMAO and/or GuHCl could be quantitatively accounted for by a model in which the free energy of dissociation decreased linearly with increasing concentration of GuHCl and increased linearly with increasing concentration of TMAO. The compensating effects of the two cosolutes in mixtures were independent of each other.

CG-SLS and DLS were used in conjunction with measurements of sedimentation velocity and fluorescence correlation spectroscopy to characterize the effect of potassium upon the self-assembly of FtsZ [96]. Whereas it was previously shown that KCl inhibits the self-association of FtsZ when bound to guanine diphosphate (FtsZ- GDP), it was found that KCl non only facilitates the self-association of FtsZ when bound to guanine triphosphate (FtsZ-GTP) but also increases the size of the large oligomeric species of FtsZ formed at sufficiently high protein concentration. In contrast, addition of KCl decreases the size of the large oligomeric species of FtsZ formed at sufficiently high concentrations of FtsZ binding a slowly hydrolyzing analog of GTP.

CG-MALS was used to determine the stoichiometry and equilibrium association constants for formation of hetero-complexes formed by the proteins αSNAP and either SNARE or V7-SNARE [97]. Under the conditions of measurement, SNARE could bind up to 4 molecules of αSNAP, and V7-SNARE could bind up to 2 molecules of αSNAP.