Nanovesicle trapping for studying weak protein interactions by single-molecule FRET

Abstract

Protein-protein interactions are fundamental biological processes. While strong protein interactions are amenable to many characterization techniques including crystallography, weak protein interactions are challenging to study because of their dynamic nature. Single-molecule fluorescence resonance energy transfer (smFRET) can monitor dynamic protein interactions in real time, but are generally limited to strong interacting pairs because of the low concentrations needed for single-molecule detection. Here, we describe a nanovesicle trapping approach to enable smFRET study of weak protein interactions at high effective concentrations. We describe the experimental procedures, summarize the application in studying the weak interactions between intracellular copper transporters, and detail the single-molecule kinetic analysis of bimolecular interactions involving three states. Both the experimental approach and the theoretical analysis are generally applicable to studying many other biological processes at the single-molecule level.

Figure 1

Schematics of nanovesicle trapping of two proteins labeled with a FRET donor-acceptor pair for smFRET studies.

Figure 2

Dependence of the effective concentration of a single molecule on the diameter of the nanovesicle. The solid symbols indicate a few commercial available membrane pore sizes for preparing nanovesicles.

Figure 3

SmFRET control experiments for acceptor blinked/bleached states and the dissociated state. (A) Two-color fluorescence intensity trajectories of a nanovesicle containing a single Cy3 molecule using 532-nm laser excitation. The Cy3 molecule photobleaches at the ~62th second. (B) Two-color fluorescence intensity trajectories of a nanovesicle containing a single Cy3 and a single Cy5. The 532-nm laser is on throughout; the 637-nm laser was turned on at the ~75th second. The Cy3 photobleaches at the ~25th second; the Cy5 molecule photobleaches at ~125th second. The first 25 seconds mimics the dissociated state of a Cy3-Cy5 pair. (C) Histograms of the apparent E_FRET (=I_A/(I_A+I_D); I_D and I_A are the fluorescence intensities of the donor and acceptor, respectively) for nanovesicles containing a single Cy3 (line patterned columns) and for nanovesicles containing a free Cy3 and a free Cy5 molecule (clear columns).

Figure 4

SmFRET measurements of weak protein interaction dynamics in a nanovesicle. (A) Two-color fluorescence intensity (upper) and corresponding apparent E_FRET (lower) trajectories of a Cy5-Hah1 and a Cy3-MBD4 trapped in a 100-nm nanovesicle. (B) Interaction scheme between Hah1 and MBD4. (C–H) Distributions of the six types of dwell times from the E_FRET trajectories of Hah1–MBD4 interactions. Solid lines are exponential fits; insets give the exponential decay constants and their relations to the protein interaction rate constants in (B). [P] is the effective concentration (~3 μM) of a single molecule in a 100-nm vesicle. The individual rate constants are: k₁ = (1.6 ± 0.2) × 10⁵ M⁻¹s⁻¹, k₋₁ = 0.88 ± 0.04 s⁻¹, k₂ = (1.4 ± 0.2) × 10⁵ M⁻¹s⁻¹, k₋₂ = 1.3 ± 0.1 s⁻¹, k₃ = 0.42 ± 0.04 s⁻¹, and k₋₃ = 0.7 ± 0.1 s⁻¹. Data in (A, C–H) adapted with permission from reference (Benitez et al., 2008; Benitez et al., 2009); Copyright 2008 American Chemical Society.

Figure 5

Generic kinetic scheme of protein interactions and corresponding E_FRET trajectories. (A) Generalized kinetic scheme of a single interacting pair with three FRET states: one dissociated state, A + A′, with a FRET value of E₀; and two interaction complexes, B and C, with FRET values of E₁ and E₂, respectively. (B) Idealized three-state E_FRET trajectories of an interacting pair; all six types of dwell times are denoted.

Scheme 1

Kinetic processes occurring during the dwell time τ₀ at the E₀ state.