Amino Acids Stabilizing Effect on Protein and Colloidal Dispersions

Enhancing protein stability with amino acids

A recent study revealed that Amino Acids (AAs) exhibit a broad colloidal stabilizing effect, impacting the dispersion of proteins, plasmid DNA, and non-biological nanoparticles. The interactions between colloidal particles were assessed using the second osmotic virial coefficient (B22) and the potential of mean force. The study suggests that charged AAs stabilize proteins of opposite charge, short peptides are as effective as multiple AAs, and any weakly interacting small molecule that enhances solvation can stabilize colloids. Experiments performed using CrystalBreeder show that AAs can shift the cloud point of lysozyme solutions by up to 4 K.

About the study

Amino acids are widely used in protein-based medical formulations to prevent aggregation and denaturation. Despite their widespread use, the nature of AAs' stabilizing effect is not fully understood, and it is unclear whether this effect is biological or a general colloidal property. This study investigates the stabilizing effect of AAs on various colloidal dispersions, proposing a new theoretical framework.

The study was carried by a team of researchers from: Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland; Southern University of Science and Technology, China; and Massachusetts Institute of Technology (MIT), USA.

Experiment Procedure

To explore the colloidal stabilizing effect of AAs, the second osmotic virial coefficient (B22) and the potential of mean force (PMF) were measured across a range of colloidal dispersions including proteins, nanoparticles, and plasmid DNA.

The cloud point measurements of lysozyme were performed using a CrystalBreeder. Solutions of lysozyme and various AAs were prepared, and their cloud points were determined by measuring transmissivity changes as the temperature decreased. In the CrystalBreeder samples were equilibrated at 42 °C for 10 minutes, then cooled at 0.2 °C/min under nitrogen flow to prevent oxidation. The cloud point temperature was recorded when a 70% loss in transmissivity was observed. This data was used to assess the stabilizing effect of AAs on protein phase behavior.

Results

The study found that AAs increased the B22 values of various proteins, indicating more stable dispersions at low concentrations without a concentration threshold. Proline's effect on gold nanoparticles and ferritin was similar to its effect on proteins, suggesting a general colloidal stabilization rather than a protein-specific interaction. The presence of AAs raised the energy barrier in the potential of mean force, stabilizing colloidal suspensions.

Cloud Point Analysis

Cloud point measurements using CrystalBreeder revealed that amino acids can shift the phase equilibrium of lysozyme solutions. Specifically, the addition of 250 mM proline lowered the equilibrium line by up to 4K for protein concentrations below 7.0 mM. The critical temperatures (Tc) and critical lysozyme concentrations (Ccr) were determined by fitting the coexistence curves to the relevant equation, demonstrating a strong match between theoretical calculations and experimental data.

where 𝐶𝑝 is the protein concentration, 𝛽 = 0.33 is the critical exponent, 𝛼, 𝑇𝑐𝑟, and 𝐶𝑐𝑟 are the adjustable parameters.  Using the cloud point equation above to fit the experimental data, shown in Figure below, found that the critical temperature 𝑇𝑐𝑟 and the critical concentration 𝐶𝑐𝑟 to be 22.9 °C and 15.3 mM in 50 mM phosphate buffer, while they become 17.1 °C and 14.6 mM, respectively, upon the addition of 250 mM proline.

Conclusion

This study shows that amino acids have a broad colloidal stabilizing effect on both biological and non-biological dispersions through weak interactions that enhance solvation and reduce attractive interactions. The CrystalBreeder was crucial for cloud point measurements, demonstrating that 250 mM proline lowered the phase equilibrium line of lysozyme solutions by up to 4K. Theoretical models accurately predicted these effects, emphasizing the importance of small molecule concentrations in colloidal formulations for improving protein stability and understanding stabilization mechanisms.

References

The research paper discussed above was authored by the following research team:

• Ting Mao, Xufeng Xu, Pamina M. Winkler, Cécilia Siri, Ekaterina Poliukhina, Paulo Jacob Silva, Quy Ong, Francesco Stellacci - Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
• Zhi Luo - Southern University of Science and Technology, Shenzhen, China
• Alfredo-Alexander Katz - Massachusetts Institute of Technology (MIT), Cambridge (MA), USA

The full paper is published here: https://arxiv.org/abs/2404.11574