Buffer Choice and pH Strongly Influence Phase Separation of SARS-CoV-2 Nucleocapsid with RNA

Mol Biol Cell. 2024 Apr 3:mbcE23120500. doi: 10.1091/mbc.E23-12-0500. Online ahead of print.ABSTRACTThe SARS-CoV-2 nucleocapsid (N) protein is crucial for virus replication and genome packaging. N protein forms biomolecular condensates both in vitro and in vivo in a process known as liquid-liquid phase separation (LLPS), but the exact factors regulating LLPS of N protein are not fully understood. Here, we show that pH and buffer choice have a profound impact on LLPS of N protein. The degree of phase separation is highly dependent on the pH of the solution, which is correlated with histidine protonation in N protein. Specifically, we demonstrate that protonation of H356 is essential for LLPS in phosphate buffer. Moreover, electrostatic interactions of buffer molecules with specific amino acid residues are able to alter the net charge of N protein, thus influencing its ability to undergo phase separation in the presence of RNA. Overall, these findings reveal that even subtle changes in amino acid protonation or surface charge caused by the pH and buffer system can strongly influence the LLPS behaviour, and point to electrostatic interactions as the main driving forces of N protein phase separation. Further, our findings emphasize the importance of these experimental parameters when studying phase separation of biomolecules, especially in the context of viral infections where the intracellular milieu undergoes drastic changes and intracellular pH normally decreases.PMID:38568799...

Source: Mol Biol Cell - April 3, 2024 Category: Molecular Biology Authors: Nina C Kathe Mihajlo Novakovic Fr édéric H-T Allain Source Type: research