The envelope (E) protein is a small polypeptide that can form ion channels in coronaviruses. In SARS coronavirus 2 (SARS-CoV-2), the agent that caused the recent COVID-19 pandemic, and its predecessor SARS-CoV-1, E protein is found in the endoplasmic reticulum–Golgi intermediate compartment (ERGIC), where virion budding takes place. Several reports claim that E protein promotes the formation of “cation-selective channels”. However, whether this term represents specificity to certain ions (e.g., potassium or calcium) or the partial or total exclusion of anions is debatable. Herein, we discuss this claim based on the available data for SARS-CoV-1 and -2 E and on new experiments performed using the untagged full-length E protein from SARS-CoV-2 in planar lipid membranes of different types, including those that closely mimic the ERGIC membrane composition. We provide evidence that the selectivity of the E-induced channels is very mild and depends strongly on lipid environment. Thus, despite past and recent claims, we found no indication that the E protein forms cation-selective channels that prevent anion transport, and even less that E protein forms bona fide specific calcium channels. In fact, the E channel maintains its multi-ionic non-specific neutral character even in concentrated solutions of Ca²⁺ ions. Also, in contrast to previous studies, we found no evidence that SARS-CoV-2 E channel activation requires a particular voltage, high calcium concentrations or low pH, in agreement with available data from SARS-CoV-1 E. In addition, sedimentation velocity experiments suggest that the E channel population is mostly pentameric, but very dynamic and probably heterogeneous, consistent with the broad distribution of conductance values typically found in electrophysiological experiments. The latter has been explained by the presence of proteolipidic channel structures.

Human serum albumin (HSA) is a target for reactive oxygen species (ROS), and alterations of its physiological functions caused by oxidation is a current issue. In this work, the amino-acid residues Trp-214 and Lys-199, which are located at site I of HSA, were experimentally and computationally oxidized, and the effect on the binding constant with phenylbutazone was measured. HSA was submitted to two mild oxidizing reagents, taurine monochloramine (Tau-NHCl) and taurine dibromamine (Tau-NBr₂). The oxidation of Trp-214 provoked spectroscopic alterations in the protein which were consistent with the formation of N′-formylkynurenine. It was found that the oxidation of HSA by Tau-NBr₂, but not by Tau-NHCl, provoked a significant increase in the association constant with phenylbutazone. The alterations of Trp-214 and Lys-199 were modeled and simulated by changing these residues using the putative oxidation products. Based on the Amber score function, the interaction energy was measured, and it showed that, while native HSA presented an interaction energy of −21.3 kJ/mol, HSA with Trp-214 altered to N′-formylkynurenine resulted in an energy of −28.4 kJ/mol, and HSA with Lys-199 altered to its carbonylated form resulted in an energy of −33.9 kJ/mol. In summary, these experimental and theoretical findings show that oxidative alterations of amino-acid residues at site I of HSA affect its binding efficacy.

Electrostatic interactions play a key role in protein structure function. There is a large family of mesoscopic protein channels whose selectivity is mainly controlled by the protein electrostatic properties and ion specific channel interactions play a minor role. The knowledge of the charge state of the ionizable residues over a wide pH range, often summarized in their pKa, stands as the most valuable information for structure-function studies of many protein channels. However, experimental pKa determination is a difficult task, typically accomplished using Nuclear Magnetic Resonance only in a limited number of membrane proteins. Thus, the pKa calculation is the most frequently used alternative. Constant pH Molecular Dynamics (CpHMD) simulation provides arguably the most accurate pKa prediction method in proteins containing many charged residues since it captures the coupling between conformational dynamics and residue protonation. Here we study the charge state of a general diffusion porin, OmpF, in which protons exert a crucial regulation of the channel discrimination of small inorganic ions as well as antibiotic translocation. We examine the pKa prediction using different methods, with the CpHMD simulations as benchmarking, and discuss the somewhat unusual titration of several acidic residues. The most widely used pKa prediction methods, though useful for globular proteins, fail to capture the specificities of channel proteins embedded in biological membranes. This is the first attempt we know to use CpHMD to study the pH- dependent charge of a large multiionic channel (with over three hundred ionizable residues) embedded in a lipid membrane.

The protonation states of key residues play a critical role in regulating the function of membrane proteins. Also, protein channel characterization involves testing the effect of pH, membrane charge, ionic concentration, and applied potential as modulators of channel conductance, selectivity and gating. Using GROMACS Constant-pH Molecular Dynamics, here we investigate how ionic strength, externally applied voltage and structural conformation changes modulate the protonation state of the acidic residues of the OmpF channel. pKa shifts in response to increased KCl concentrations do not follow a simple pattern. This contrasts with earlier claims on the effect of ionic strength on the pKa of selected OmpF residues. Additionally, we find that applying an external voltage yields position-dependent small pKa shifts of opposite sign on the periplasmic and the extracellular side of the protein. This charge regulation coming from the external electric field adds to the well-known structural charge asymmetry of this channel. While pKa shifts involve minimal protein backbone conformation changes, there are some significant side chains rotations or displacements. Overall, these findings provide new insights into how different factors influence the protonation dynamics of OmpF. The results may have useful implications for other channels regulated by long-range electrostatic interactions