Deciphering the liquid-liquid phase separation induced modulation in the structure, dynamics, and enzymatic activity of an ordered protein β-lactoglobulin

Abstract

Owing to the significant role in the subcellular organization of biomolecules, physiology, and the realm of biomimetic materials, studies related to biomolecular condensates formed through liquid-liquid phase separation (LLPS) have emerged as a growing area of research. Despite valuable contributions of prior research, there is untapped potential in exploring the influence of phase separation on the conformational dynamics and enzymatic activities of native proteins. Herein, we investigate the LLPS of β-lactoglobulin (β-LG), a non-intrinsically disordered protein, under crowded conditions. In-depth characterization through spectroscopic and microscopic techniques revealed the formation of dynamic liquid-like droplets, distinct from protein aggregates, driven by hydrophobic interactions. Our analyses revealed that phase separation can alter structural flexibility and photophysical properties. Importantly, the phase-separated β-LG exhibited efficient enzymatic activity as an esterase; a characteristic seemingly exclusive to β-LG droplets. The droplets acted as robust catalytic crucibles, providing an ideal environment for efficient ester hydrolysis. Further investigation into the catalytic mechanism suggested the involvement of specific amino acid residues, rather than general acid or base catalysis. Also, the alteration in conformational distribution caused by phase separation unveils the latent functionality. Our study delineates the understanding of protein phase separation and insights into the diverse catalytic strategies employed by proteins. It opens exciting possibilities for designing functional artificial compartments based on phase-separated biomolecules.

Fig. 1. Experimental investigation of phase separation conditions for β-lactoglobulin (β-LG) in the presence of polyethylene glycol (PEG8000) at 37 °C and pH 7.4. (a) Molecular structures of β-LG and PEG (n = 8000). (b) Time-dependent turbidity analysis of 50 μM β-LG incubated with 10% w/v PEG8000 at 37 °C and pH 7.4. (c) Turbidity profiles of β-LG incubated with a fixed concentration of 10% w/v PEG8000 at different protein concentrations at 37 °C and pH 7.4. (d)–(f) Morphological evolution of droplets observed through FESEM studies during incubation of 50 μM β-LG with 10% w/v PEG8000 at 37 °C and pH 7.4 for 1, 3 and 7 day(s), respectively. (g)–(i) Corresponding DIC images of 50 μM β-LG incubated with 10% w/v PEG8000 at 37 °C and pH 7.4 for 1, 3 and 7 day(s), respectively.

Fig. 2. Characterization of the CPM-labelled β-LG droplets and FRAP. (a) Schematic representation of the labelling process of β-LG with CPM dye. (b)–(d) Represent confocal microscopy images (left to right: fluorescence intensity-based images, DIC images and the colocalization images) of the β-LG droplets (50 μM β-LG, with 5% CPM-labelled β-LG, in 10% w/v PEG8000, at 37 °C and pH 7.4) for day1, day3 and day7, respectively (scale bar: 3 μm). (e) Fluorescence images of β-LG droplets (50 μM β-LG, with 5% CPM-labelled, in 10% w/v PEG8000, at 37 °C and pH 7.4 for day5) (obtained by photobleaching a ROI) before bleaching, after bleaching, and post-recovery of fluorescence during the FRAP experiments (scale bar: 3 μm). (f) FRAP kinetics of phase-separated β-LG droplets (50 μM β-LG, with 5% CPM-labelled, in 10% w/v PEG8000, at 37 °C and pH 7.4), demonstrating rapid fluorescence recovery with an average half-lifetime of 60 seconds. The error bar represents mean ± standard deviation (s.d.) for n = 11 independent experiments.

Fig. 3. Characterizing protein conformation in the phase-separated state. (a) Anisotropy measurements using CPM-labelled protein for native and phase-separated states (50 μM β-LG, with 5% CPM-labelled β-LG, in 10% w/v PEG8000, at 37 °C and pH 7.4) reveal the dynamic molecular reorientation of the protein within the liquid condensates. (b) Far-UV CD spectra of 50 μM β-LG in 10% w/v PEG8000 (incubated at 37 °C and pH 7.4) recorded with a diluted solution at an effective protein concentration of 5 μM within the instrument limitation, to avoid the saturation of the detector. The dilution maintains the liquid droplet morphology unaffected (data not shown here). (c) The fluorescence spectra of β-LG undergoing the phase separation (λ_ex = 295 nm) display the gradual increase in charge transfer band intensity through FRET between the intrinsic tryptophan and the charge transfer species. (d) Charge transfer fluorescence excitation and emission spectra of the phase-separated state of β-LG centered around 320 nm and 400 nm, respectively.

Fig. 4. Investigating the effect of electrolyte and chaotropic agent on the LLPS of β-LG. (a) Turbidity plot of the β-LG (50 μM) incubated with a fixed concentration of PEG8000 (10% w/v) in the presence of different concentrations of NaCl. (b) Fluorescence image of the sample incubated with 300 mM of NaCl and (c) merged image combining fluorescence and phase-contrast images of the sample incubated with 300 mM of NaCl (scale bar: 5 μm). (d) Turbidity plot of the sample (50 μM β-LG in the presence of 10% w/v PEG8000, at 37 °C and pH 7.4) incubated with varying percentages of 1,6-hexanediol in the solution. (e) Fluorescence image of the sample incubated with 6% of 1,6-hexanediol in the solution and (f) merged image combining fluorescence and phase-contrast images of the sample incubated with 6% of 1,6-hexanediol in the solution (scale bar: 9 μm).

Fig. 5. The functional activity of the protein under native and phase-separated conditions. (a) Structural representation of the ester substrates PNPA, PNPB, and PNPV used for analysis. (b) Gradual enhancement in the absorbance peak around 400 nm for the hydrolyzed product, PNP in the presence of liquid–liquid phase-separated β-LG. (c) Esterase kinetics of PNPA hydrolysis (keeping [PNPA] = 120 μM; monitoring absorbance at 400 nm) by β-LG condensates (50 μM β-LG in 10% w/v PEG8000, at 37 °C and pH 7.4 after 7 days) compared with the native protein (50 μM), PEG8000 (10% w/v), and buffer as controls. (d) Comparative analysis of the rate of hydrolysis of PNPA as a function of its concentration by β-LG condensates, the native protein, PEG8000, and phosphate buffer. (e) Comparative analysis of the catalytic rate constant (k_cat) for PNPA hydrolysis using β-LG condensates, the native protein, PEG8000, and phosphate buffer under similar conditions (having PNPA substrate concentration of 120 μM, 50 μM β-LG, 10% w/v PEG8000). (f) Comparative analysis of the catalytic rate constant (k_cat) for different substrates, viz. 120 μM PNPA, PNPB, and PNPV under similar conditions (50 μM β-LG, 10% w/v PEG8000; temperature 37 °C and pH 7.4) using liquid–liquid phase-separated β-LG. (g) Fluorescence emission spectra of DCFDA when excited at 490 nm; the inset structure represents the chemical structure of the non-emissive DCFDA and the image represents the confocal micrograph of the droplets immediately after the addition of a fresh solution of DCFDA (scale bar: 3 μm). (h) Fluorescence emission spectra of DCF (λ_ex/em = 490/530 nm) monitored as a function of time; the inset structure represents the chemical structure of the hydrolyzed product DCF which is emissive and the image represents the confocal micrograph of the droplets (brightfield merged with the fluorescence image) after 24 h hydrolysis of DCFDA (scale bar: 3 μm). (i) Comparative analysis of the fluorescence emission of DCF formed through the different extent of hydrolysis from β-LG condensates, native protein, PEG8000, and phosphate buffer under similar conditions.