Interfacial Protein–Protein Associations

Abstract

While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface – with areas of high protein density (i.e. strongly-interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e. partial-RET (monomer-monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e. clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage.

Single-Molecule Resolution of Protein Dynamics on Polymeric Membrane Surfaces: The Roles of Spatial and Population Heterogeneity

“…For example, in previous work, transient protein−protein associations were observed to increase the surface residence time for BSA on a PEG monolayer at protein concentrations as low as 2.5 × 10 −6 mg/mL. 45 Although molecular fluorescence intensity serves as only a lower estimate of oligomerization state when unlabeled proteins are present, we saw both fluorescently labeled monomers and oligomers at high protein concentrations on the surface (see the Supporting Information). For both monomers and oligomers, we observed shorter residence times at high protein concentrations.…”

“…In previous work, we demonstrated that dynamic protein−protein interfacial associations occurred at solution concentrations as low as 2.5 × 10 −6 mg/mL for BSA on a PEG monolayer (at a surface coverage of 2.5 molecules per μm 2 ). 45 In the experiments described in this section, protein solution concentrations were increased to 0.3 mg/mL for IgG and 1.0 mg/mL for BSA by mixing unlabeled protein and low concentrations of fluorescently labeled protein (10 −5 −10 −6 mg/mL). Labeled proteins served as reporter molecules of interfacial dynamics at these high protein concentrations.…”

Macromolecular Crowding In Vitro , In Vivo , and In Between

“…It is however necessary to recognize that all differences between specified biochemical equilibria and rates within an intact cell and the same equilibria and rates in a dilute solution of purified macromolecules cannot and should not be attributed to crowding. One cannot ignore the likelihood of additional modulating effects deriving from interactions of soluble macromolecules with membranes and other structural elements 6–9 , in addition to unidentified or incompletely characterized interactions between the studied reactants and other soluble species such as molecular chaperones 10 . Thus we would like to make clear at the outset that the term macromolecular crowding should be used to describe only those effects arising from the presence of a total high weight per volume concentration of functionally unrelated soluble macromolecules.…”

Capturing Conformation-Dependent Molecule–Surface Interactions When Surface Chemistry Is Heterogeneous

“…86 Due to the significant uncertainty in the parameter μ and the absolute fluorescence intensities, we reported our data using the relative end-to-end distance d = r/μ = (F D /F A ) 1/6 as in previous single-molecule FRET studies of freely adsorbing molecules. 54,55,83,87 Calculation of Likelihood of Conformational States. For a data set with N total observations, the likelihood of observing a helix is n(helix)/N, where n(helix) is the number of observations of the helix state.…”