Isoelectric Points of Proteins at the Air/Liquid Interface and in Solution.

Electrostatic interactions play essential roles in determining the function, colloidal stability, and adsorption of proteins on different surfaces and interfaces. Therefore, a molecular-level understanding of the charge state of the proteins under different conditions is required to explain their macroscopic properties. In this study, we have employed an inherently surface-sensitive spectroscopic tool, sum frequency generation spectroscopy, to determine the charge state of a wide range of proteins as a function of pH at the air/liquid interface via measurement of the degree of orientation of water molecules. We compared the isoelectric point (IEP) of the 12 investigated proteins at the air/liquid interface with that in the bulk solution obtained through zeta potential measurements. Ellipsometry is performed to determine the film thickness at the air/liquid interface at different charge states. In particular, protein aggregation at the IEP is reflected by increased film thickness. For all proteins, the interfacial point of zero charge is close (with less than 1 pH unit variation) to that in the bulk solution.

A Comprehensive Brownian Dynamics Approach for the Determination of Non-ideality Parameters from Analytical Ultracentrifugation.

Brownian dynamics (BD) has been applied as a comprehensive tool to model sedimentation and diffusion of nanoparticles in analytical ultracentrifugation (AUC) experiments. In this article, we extend the BD algorithm by considering space-dependent diffusion and solvent compressibility. With this, the changes in the sedimentation and diffusion coefficient from altered solvent properties at increased pressures are accurately taken into account. Moreover, it is demonstrated how the concept of space-dependent diffusion is employed to describe concentration-dependent sedimentation and diffusion coefficients, in particular, through the Gralen coefficient and the second virial coefficient. The influence of thermodynamic nonideality on diffusional properties can be accurately simulated and agree with well-known evaluation tools. BD simulations for sedimentation equilibrium and sedimentation velocity (SV) AUC experiments including effects of hydrodynamic and thermodynamic nonideality are validated by global evaluation in SEDANAL. The interplay of solvent compressibility and retrieved nonideality parameters can be studied utilizing BD. Finally, the second virial coefficient is determined for lysozyme from SV AUC experiments and BD simulations and compared to membrane osmometry. These results are in line with DLVO theory. In summary, BD simulations are established for the validation of nonideal sedimentation in AUC providing a sound basis for the evaluation of complex interactions even in polydisperse systems.

Effect of pH and urea on the proteins secondary structure at the water/air interface and in solution.

HYPOTHESIS: The secondary structure of proteins affects their functionality and performance in physiological environments or industrial applications. Change of the solution pH or the presence of protein denaturants are the main chemical means that can alter the secondary structure of proteins or lead to protein denaturation. Since proteins in the bulk solution and those residing at the solution/air interface experience different local environments, their response to chemical denaturation can be different. EXPERIMENTS: We utilize circular dichroism and chiral/achiral sum frequency generation spectroscopy to study the secondary structure of selected proteins as a function of the solution pH or in the presence of 8 M urea in the bulk solution and at the solution/air interface, respectively. FINDINGS: The liquid/air interface can enhance or decrease protein conformation stability. The change in the secondary structure of the surface adsorbed proteins in alkaline solutions occurs at pH values lower than those denaturing the studied proteins in the bulk solution. In contrast, while 8 M urea completely denatures the studied proteins in the bulk solution, the liquid/air interface prevents the urea-induced denaturation of the surface adsorbed proteins by limiting the access of urea to the hydrophobic side chains of proteins protruding to air.