Protein folding, binding, and droplet formation in cell-like conditions

Abstract

The many bystander macromolecules in the crowded cellular environments present both steric repulsion and weak attraction to proteins undergoing folding or binding and hence impact the thermodynamic and kinetic properties of these processes. The weak but nonrandom binding with bystander macromolecules may facilitate subcellular localization and biological function. Weak binding also leads to the emergence of a protein-rich droplet phase, which has been implicated in regulating a variety of cellular functions. All these important problems can now be addressed by realistic modeling of intermolecular interactions. Configurational sampling of concentrated protein solutions is an ongoing challenge.

Fig. 1

Protein folding, binding, and droplet formation inside a cell. “Test” proteins are volume-excluded from but also weakly bind to bystander macromolecules in the cellular environment, and these interactions can steer folding and binding stability in complex ways. Test proteins (either unstructured or structured) can also weakly interact among themselves, and form a new, droplet phase in the cellular environment.

Fig. 2

Direct simulation versus postprocessing approach, illustrated on the folding of cytochrome b₅₆₂. (a) Direct simulation follows the vertical paths, whereas postprocessing follows the vertical paths. The former approach yields the folding free energies in the absence (ΔG_f0) and presence (ΔG_f) of crowders, whereas the latter approach yields the transfer free energies of the unfolded (Δμ_U) and folded (Δμ_F) states from a dilute solution to the crowder solution. By closing a thermodynamic cycle, they lead to the same effect of crowding on the folding free energy, ΔΔG_f. Taken from ref [38]. (b) To calculate Δμ(X), one has to fictitiously place the protein with conformation X into different positions and evaluate the protein-crowder interaction energy at each position. Taken from ref [5].

Fig. 3

Nonrandom weak binding of the maltose-binding protein (MBP) and the Pin1 WW domain with bystander macromolecules. (a) Competition of Ficoll and maltose for interaction with MBP, shown by NMR spectroscopy. In buffer, apo MBP shows well-resolved ¹H-¹⁵N TROSY spectra. With 200 g/l Ficoll, most of the TROSY peaks are broadened beyond detection, indicating MBP-Ficoll binding. Upon further addition of 1 mM maltose, the peaks are recovered, indicating that the ligand has competed out the weakly bound Ficoll. (b) Shuttling of MBP in the E. coli periplasm for transport of maltose into the cytoplasm. The apo form may be weakly bound to the outer membrane-attached peptidoglycan; upon binding maltose, MBP is released from the peptidoglycan and diffuses toward the inner membrane, where it hands over the ligand to the ABC transporter for translocation into the cytoplasm. Red and black arrows indicate the flow of maltose and the shuttling of MBP, respectively. (a)and (b) taken from [12]. (c) Protein-crowder interaction energies calculated by FMAP. Top panel: the test protein (green) is the Pin1 WW protein, and the crowder is ovalbumin, with 8 copies present in a cubic box with a 157.4-Å side length (corresponding to a concentration of approximately 150 mg/mL). The crowder configuration was a snapshot taken from molecular dynamics simulation in explicit solvent. Note that the crowder molecules formed clusters. Bottom panel: in the FMAP calculation, both the protein and crowder molecules were represented at the all-atom level, and the energy function consisted of Lennard-Jones terms for modeling steric, van der Waals, and hydrophobic interactions and Debye-Hu ckel terms for modeling electrostatic interactions [73]. The energy map on a slice through the crowder box is shown according to a color scale from white to dark red; the gray regions are occupied by the crowder molecules. The placement of the test protein shown in the top panel has the minimum interaction energy, in which the substrate recognition site of the WW domain forms close contacts with one of the ovalbumin molecules (enlarged view on the left).

Fig. 4

Calculation of liquid-liquid coexistence curves [23]. (a) By the Widom insertion, such as implemented by FMAP, the chemical potentials of a protein over a range of concentrations are obtained. (b) Left: the phase co-existence condition is located by applying the Maxwell equal-area rule on the isotherm in the chemical potential (μ) – concentration (ρ) plane. The blue horizontal dash (at μ = μ_CO) crosses the isotherm with equal areas enclosed above and below. The concentrations (ρ₁ and ρ₂) at the low and high crossing points are those of the dissolved and droplet phases, respectively. Right: by repeating the process over a range of temperature, the full phase diagram is constructed. (c) Liquid-liquid phase diagram for γII-crystallin calculated by FMAP, compared to the experimental data. In the calculation, γII-crystallin molecules were represented at the all-atom level, and their interactions were modeled by Lennard-Jones and Debye-Hu ckel potentials. Snapshots of protein configurations at 123 and 307 mg/mL in an 81-Å thick slab are shown to the left and right, respectively.