Proteins that encounter unfavorable solvent conditions are prone to aggregation, a phenomenon that remains poorly understood. This work focuses on myoglobin (Mb) as a model protein. Upon heating, Mb produces amorphous aggregates. Thermal unfolding experiments at low concentration (where aggregation is negligible), along with centrifugation assays, imply that Mb aggregation proceeds via globally unfolded conformers. This contrasts studies on other proteins that emphasized the role of partially folded structures as aggregate precursors. Molecular dynamics (MD) simulations were performed to gain insights into the mechanism by which heat-unfolded Mb molecules associate with one another. A prerequisite for these simulations was the development of a method for generating monomeric starting structures. Periodic boundary condition artifacts necessitated the implementation of a partially immobilized water layer lining the walls of the simulation box. Aggregation simulations were performed at 370 K to track the assembly of monomeric Mb into pentameric species. Binding events were preceded by multiple unsuccessful encounters. Even after association, protein−protein contacts remained in flux. Binding was mediated by hydrophobic contacts, along with salt bridges that involved hydrophobically embedded Lys residues. Overall, this work illustrates that atomistic MD simulations are well suited for garnering insights into protein aggregation mechanisms.

Many aspects of protein function rely on conformational fluctuations. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) provides a window into these dynamics. Despite the widespread use of HDX-MS, it remains unclear whether this technique provides a truly comprehensive view of protein dynamics. HDX is mediated by H-bond-opening/closing events, implying that HDX methods provide an H-bond-centric view. This raises the question if there could be fluctuations that leave the H-bond network unaffected, thereby rendering them undetectable by HDX-MS. We explore this issue in experiments on cytochrome c (cyt c). Compared to the Fe(II) protein, Fe(III) cyt c shows enhanced deuteration on both the distal and proximal sides of the heme. Previous studies have attributed the enhanced dynamics of Fe(III) cyt c to the facile and reversible rupture of the distal M80−Fe(III) bond. Using molecular dynamics (MD) simulations, we conducted a detailed analysis of various cyt c conformers. Our MD data confirm that rupture of the M80−Fe(III) contact triggers major reorientation of the distal Ω loop. Surprisingly, this event takes place with only miniscule H-bonding alterations. In other words, the distal loop dynamics are almost "HDX-silent". Moreover, distal loop movements cannot account for enhanced dynamics on the opposite (proximal) side of the heme. Instead, enhanced deuteration of Fe(III) cyt c is attributed to sparsely populated conformers where both the distal (M80) and proximal (H18) coordination bonds have been ruptured, along with opening of numerous H-bonds on both sides of the heme. We conclude that there can be major structural fluctuations that are only weakly coupled to changes in H-bonding, making them virtually impossible to track by HDX-MS. In such cases, HDX-MS may provide an incomplete view of protein dynamics.

Highlights A carbohydrate blend improved acceptability of lobster meat frozen for one-year. There was minimal loss in moisture in lobster meat frozen in the carbohydrate blend. Increased tenderness was observed in lobster meat frozen in the carbohydrate blend.

Peptide separations by reversed-phase liquid chromatography (RPLC) are an integral part of bottom-up proteomics. These separations typically employ C18 columns with water/acetonitrile gradient elution in the presence of formic acid. Despite the widespread use of such workflows, the exact nature of peptide interactions with the stationary and mobile phases is poorly understood. Here, we employ microsecond molecular dynamics (MD) simulations to uncover details of peptide RPLC. We examined two tryptic peptides, a hydrophobic and a hydrophilic species, in a slit pore lined with C18 chains that were grafted onto SiO 2 support. Our simulations explored peptide trapping, followed by desorption and elution. Trapping in an aqueous mobile phase was initiated by C18 contacts with Lys butyl moieties. This was followed by extensive anchoring of nonpolar side chains (Leu/Ile/Val) in the C18 layer. Exposure to water/acetonitrile triggered peptide desorption in a stepwise fashion; charged sites close to the termini were the first to lift off, followed by the other residues. During water/acetonitrile elution, both peptides preferentially resided close to the pore center. The hydrophilic peptide exhibited no contacts with the stationary phase under these conditions. In contrast, the hydrophobic species underwent multiple transient Leu/Ile/Val binding interactions with C18 chains. These nonpolar interactions represent the foundation of differential peptide retention, in agreement with the experimental elution behavior of the two peptides. Extensive peptide/formate ion pairing was observed in water/acetonitrile, particularly at N-terminal sites. Overall, this work uncovers an unprecedented level of RPLC molecular details, paving the way for MD simulations as a future tool for improving retention prediction algorithms and for the design of novel column materials.