Application

DLS can be used to rank molecules for their propensity to self-aggregate.Two parameters (kD and A2) can be negative (self-association), neutral (no interaction) or positive (repulsive forces).

Protein intrinsic fluorescence and isothermal chemical denaturation (ICD). (A) The use of intrinsic fluorescence detection is the most powerful for proteins with tryptophan moieties with the best fluorescence properties, followed by tyrosine and phenylalanine, which limit not only the quantum yield, but also low-wavelength emission and excitation. Red shift in tryptophan fluorescence typically occurs during protein denaturation and its fluorescence is also affected by nearby tyrosine. (B) ICD is performed in a selected temperature, typically RT or 37 °C, performing denaturant titration with each sample and monitoring the signal at equilibrium. Often, chemical denaturation is reversible, unlike temperature-induced unfolding, which in many cases is irreversible.

Folding and unfolding of β-lactoglobulin in 2.0 M urea. (A) Model-independent evaluation of the heat capacity Cp(T). (B) Enthalpy ΔH(T). Black data points: integration of Cp(T). Red data points: black data points shifted downwards by 78.3 kcal/mol, the enthalpy of cold denaturation. (C) Entropy ΔS(T). Black: integration of Cp(T). Red: primary data points shifted downwards by 0.283 kcal/molK, the unfolding entropy of cold denaturation. (D) Gibbs free energy ΔG(T). Black: application of △G equation to black data points in panels B and C. Red: combination of red data points in panels B and C, according to △G equation.

Effect of solvent, cosolvent, and additive on catalytic activity of human BChE submitted to high pressure. Loss in activity of human BChE in 10 mM Tris/HCl, pH 7.4 at 25 °C, as a function of hydrostatic pressure in the absence of a cosolvent (blue curve), in the presence of a stabilizer additive (red curve), and in the presence of a destabilizing cosolvent (green curve). Cosolvent (20% v/v) and stabilizer (20% w/v) lead to shifts in P1/2, the pressure causing 50% of enzyme inactivation.

Spectral Center of Mass Analysis

Spectral center of mass (COM, or barycentric mean, BCM) obtained for aCgn 0.025 mg/mL (A) and Ova 0.3mg/mL (B) as function of increasing pressure for a series of T. Inset shows fluorescence curves obtained at 3 kbar for a series of T. (20℃, green; -5℃, grey; -10℃, light blue; -14℃, dark blue). Arrows represent the decrease of T at 3 kbar. COM profiles are also shown as a function of denaturant concentration (purple circles) as a reference for a complete folded to unfolded transition: urea and guanidine were used to unfold aCgn and Ova respectively. COM/BCM was calculated using intrinsic fluorescence profiles.

Uses of DSF to study proteins and their interactions. (a) Ligand binding to a protein will typically stabilize the system such that the protein–ligand complex will have a higher T_ma value. (b) In the corresponding DSF plot, ligand binding shifts the curve to the right, providing a ΔT_ma value that is indicative of binding. In these experiments, it is important to include a systematic series of control experiments to identify potential artifacts. In each control, a component of the system (e.g., protein, dye, and ligand) is removed. In the idealized case shown, the low fluorescence of each control (dotted lines) suggest that the measured T_ma values are accurate. DSF can also be used to explore the effects of (c) mutations, (d) buffer components (e.g., divalent cation, detergents), or (e) protein–protein interactions (PPIs) on stability.