Mechanism of Thermal Protein Aggregation: Experiments and Molecular Dynamics Simulations on the High-Temperature Behavior of Myoglobin

Abstract

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.

Atomistic Details of Peptide Reversed-Phase Liquid Chromatography from Molecular Dynamics Simulations

“…However, Lys/C18 contacts only involved the aliphatic region of the −CH 2 –CH 2 –CH 2 –CH 2 –NH 3 + side chains, while the charged amino groups remained in water contact (Figure C,H). It has been demonstrated previously that Lys can engage in hydrophobic contacts via its aliphatic region, for example, during protein aggregation . The Lys/C18 contacts seen here are consistent with those earlier data.…”

“…It has been demonstrated previously that Lys can engage in hydrophobic contacts via its aliphatic region, for example, during protein aggregation. 76 The Lys/C18 contacts seen here are consistent with those earlier data.…”

Structural Dynamics of a Thermally Stressed Monoclonal Antibody Characterized by Temperature-Dependent H/D Exchange Mass Spectrometry

“…In such a case, backbone amides in the thermally unfolded moieties would be completely unprotected, akin to amides in dipeptide model compounds . However, it is known that many thermally unfolded proteins possess residual protection, suggesting that globally mediated HDX requires two steps (N ⇄ U closed ⇄ U open ), where the second step produces H open sites that are required for deuteration. ,− Within our thermodynamic framework, such two-step transitions are captured via eq , where Δ G glob ( T ) refers to the N ⇄ U closed transitions that can be probed by DSC, while Δ G opU refers to U closed ⇄ U open Figure F shows that many CH2 and CH3 segments had Δ G opU values around 20 – 50 kJ mol –1 , revealing that HDX of the corresponding segments indeed proceeds via N ⇄ U closed ⇄ U open .…”

“…To enhance the dynamic range of our experiments, the data discussed below were recorded using slightly more acidic solutions, 46,50 i.e., pH meter reading of 6.3 (corresponding to pD 6.7). 69 HDX was performed at 0, 23,30,40,50,55,60,65,70,75,80,85,90, and 95 °C in Eppendorf tubes that were immersed in a Tcontrolled water bath. Prior to HDX, D 2 O labeling buffer was preequilibrated at the desired temperature, while NISTmAb was kept at room temperature to avoid aggregation of the stock solution.…”

“…In eq , Δ G glob ( T ) is the free energy of global unfolding that can be measured by DSC . The Δ G opU > 0 term in eq accounts for residual protection of the unfolded state, i.e., the fact that NH sites in unfolded proteins exchange more slowly than in truly unprotected model peptides. ,− In summary, eqs – provide a model that links the T dependence of K op and k ch to fundamental thermodynamic parameters, while separating the effects of local and global dynamics. This detailed analysis goes beyond earlier studies, , where T -dependent changes of k ch were compensated by simply altering the labeling time, without deciphering the combined effects of local and global dynamics on K op ( T ).…”