H/D exchange (HDX) mass spectrometry (MS) is a widely used technique for interrogating protein structure and dynamics. Backbone HDX is mediated by opening/closing (unfolding/refolding) fluctuations. In traditional HDX–MS, proteins are incubated in D2O as a function of time at constant temperature (T). There is an urgent need to complement this traditional approach with experiments that probe proteins in a T-dependent fashion, e.g., for assessing the stability of therapeutic antibodies. A key problem with such studies is the absence of strategies for interpreting HDX–MS data in the context of T-dependent protein dynamics. Specifically, it has not been possible thus far to separate T-induced changes of the chemical labeling step (k ch) from thermally enhanced protein fluctuations. Focusing on myoglobin, the current work solves this problem by dissecting T-dependent HDX–MS profiles into contributions from k ch(T), as well as local and global protein dynamics. Experimental profiles started off with surprisingly shallow slopes that seemed to defy the quasi-exponential k ch(T) dependence. Just below the melting temperature (T m) the profiles showed a sharp increase. Our analysis revealed that local dynamics dominate at low T, while global events become prevalent closer to T m. About half of the backbone NH sites exhibited a canonical scenario, where local opening/closing was associated with positive ΔH and ΔS. Many of the remaining sites had negative ΔH and ΔS, thereby accounting for the shallowness of the experimental HDX–MS profiles at low T. In summary, this work provides practitioners with the tools to analyze proteins over a wide temperature range, paving the way toward T-dependent high-throughput screening applications by HDX–MS.

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.

Cu(I) nanoparticles (NPs) on modified poly(styrene-co-maleic anhydride) was prepared via a facile procedure. In this regard the modified co-polymer (SMA) was initially synthesized from the reaction of poly(styrene-co-maleic anhydride) with 3-aminopyridine. Upon the treatment of CuI with SMA, Cu(I) NPs were immobilized on SMA. This immobilized Cu(I) NPs was fully characterized by FTIR, SEM and EDAX analysis methods. Moreover, the interaction of Cu cations with 3-aminopyridine modified SMI catalyst via density functional theory (DFT) and quantum theory of atoms in molecules (QTAIM) computationally was studied. Our calculated results demonstrated that among 2,3 and 4 substituted aminopyridines for the modification of SMA, the 3-amnopyridine has the weakest ligand and Cu(I) interaction. However, the SMI image showed a suitable size of nanoparticles and inductively coupled plasma (ICP) analysis showed a higher Cu content in comparison with those of 2 and 4-substituted pyridines. The catalytic activity and reusability of this nanocatalyst system of 3-substituded aminopyridines was examined in the Huisgen 1,3-dipolar cycloaddition via click reaction. The reactions proceeded smoothly leading to regioselective synthesis of 1,4-disubstitued 1,2,4-triazole derivatives in excellent yields under mild reaction conditions. This heterogeneous catalytic system was separated easily by simple filtration and was reused without pre-activation at least in five runs without appreciable loss in its activity.

Differential scanning calorimetry (DSC) is a standard tool for probing the resilience of monoclonal antibodies (mAbs) and other protein therapeutics against thermal degradation. Unfortunately, DSC usually only provides insights into global unfolding, although sequential steps are sometimes discernible for multidomain proteins. Temperature-dependent hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has the potential to probe heat-induced events at a much greater level of detail. We recently proposed a strategy to deconvolute temperature-dependent HDX data into contributions from local dynamics, global unfolding/refolding, as well as chemical labeling. However, that strategy was validated only for a small protein (Tajoddin, N. N.; Konermann, L. Anal. Chem. 2020, 92, 10058). The current work explores the applicability of this HDX framework to the NIST reference mAb (NISTmAb), a large multidomain protein that is prone to aggregation and has three melting points. Using global fitting, we were able to model HDX profiles across the NISTmAb sequence between zero and 95 °C, and for time points between 15 s and 20 min. We uncovered the enthalpic and entropic contributions of local fluctuations that govern the conformational dynamics at low temperatures. The CH2 and CH3 domains were found to be increasingly affected by global unfolding/refolding in the vicinity of their melting points, although the transiently unfolded protein displayed significant residual protection. Global dynamics were not involved in the deuteration of the Fab domains (which have the highest melting point). Instead, global Fab unfolding was followed immediately by irreversible aggregation. Our results reveal that the thermodynamic HDX-MS strategy applied in this work is well suited for probing spatially resolved dynamics of thermally stressed large proteins such as mAbs, complementing data obtained by DSC.