Deciphering the Thermal Stability of Bacteriophage MS2-Derived Virus-like Particle and Its Engineered Variant.

RNA bacteriophage MS2-derived virus-like particles (VLPs) have been widely used in biomedical research as model systems to study virus assembly, structure-function relationships, vaccine development, and drug delivery. Considering the diverse utility of these VLPs, a systemic engineering approach has been utilized to generate smaller particles with optimal serum stability and tissue penetrance. Additionally, it is crucial to demonstrate the overall stability of these mini MS2 VLPs, ensuring cargo protection until they reach their target cell/organ. However, no detailed analysis of the thermal stability and heat-induced disassembly of MS2 VLPs has yet been attempted. In this work, we investigated the thermal stability of both wild-type (WT) MS2 VLP and its "mini" variant containing S37P mutation (mini MS2 VLP). The mini MS2 VLP exhibits a higher capsid melting temperature (Tm) when compared to its WT MS2 VLP counterpart, possibly attributed to its smaller interdimer angle. Our study presents that the thermal unfolding of MS2 VLPs follows a sequential process involving particle destabilization, nucleic acid exposure/melting, and disassembly of VLP. This observation underscores the disruption of cooperative intersubunit interactions and protein-nucleic acid interactions, shedding light on the mechanism of heat-induced VLP disassembly.

Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

The Cryo-EM STRUCTURE of Renal Amyloid Fibril Suggests Structurally Homogeneous Multiorgan Aggregation in AL Amyloidosis.

Immunoglobulin light chain amyloidosis (AL) is caused by the aberrant production of amyloidogenic light chains (LC) that accumulate as amyloid deposits in vital organs. Distinct LC sequences in each patient yield distinct amyloid structures. However different tissue microenvironments may also cause identical protein precursors to adopt distinct amyloid structures. To address the impact of the tissue environment on the structural polymorphism of amyloids, we extracted fibrils from the kidney of an AL patient (AL55) whose cardiac amyloid structure was previously determined by our group. Here we show that the 4.0 Å resolution cryo-EM structure of the renal fibril is virtually identical to that reported for the cardiac fibril. These results provide the first structural evidence that LC amyloids independently deposited in different organs of the same AL patient share a common fold.

Elucidation of the folding pathway of a circular permutant of topologically knotted YbeA by tryptophan substitutions.

CP74 is an engineered circular permutant of a deep trefoil knotted SpoU-TrmD (SPOUT) RNA methyl transferase protein YbeA from E. coli. We have previously established that the circular permutation unties the knotted topology of YbeA and CP74 forms a domain-swapped dimer with a large dimeric interface of ca. 4600 Å2. To understand the impact of domain-swapping and the newly formed hinge region joining the two folded domains on the folding and stability of CP74, the five equally spaced tryptophan residues were individually substituted into phenylalanine to monitor their conformational and stability changes by a battery of biophysical tools. Far-UV circular dichroism, intrinsic fluorescence, and small-angle X-ray scattering dictated minimal global conformational perturbations to the native structures in the tryptophan variants. The structures of the tryptophan variants also showed the conservation of the domain-swapped ternary structure with the exception that the W72F exhibited significant asymmetry in the α-helix 5. Comparative global thermal and chemical stability analyses indicated the pivotal role of W100 in the folding of CP74 followed by W19 and W72. Solution-state NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry further revealed the accumulation of a native-like intermediate state in which the hinge region made important contributions to maintain the domain-swapped ternary structure of CP74.

Elucidation of folding pathways of knotted proteins.

Understanding the mechanisms by which proteins fold and thread into topologically knotted conformations has been challenging because of the apparent complexity associated with the folding and threading events. Nevertheless, many experimental and computational studies have provided insights into the folding pathways of knotted proteins and showed that most of the knotted proteins could spontaneously and reversibly fold into knotted topologies with highly populated intermediates and, at times, through multiple folding pathways. Our laboratory has reported the folding mechanisms of a variety of knotted proteins that have different knot types, ranging from the simplest trefoil 31 knot to the most complex Stevedore's 61 knot. Therefore, we focused on using multiplex thermodynamics and kinetics measurements to tease out unique information associated with different structural probes to obtain a more comprehensive overview of the folding mechanisms of the knotted proteins of interest. In this chapter, we shall discuss the use of different biophysical tools and analytical models to glean mechanistic insights into how intricate polypeptides attain knotted topologies.

Impacts of Cancer-associated Mutations on the Structure-Activity Relationship of BAP1.

BRAC1 associated protein-1 (BAP1) is a major tumor suppressor involved in many cancers. The deubiquitinase (DUB) activity of BAP1 is essential for its nuclear localization, histone remodeling and proteostasis associated with mitochondrial calcium flux. Loss of the DUB activity due to catalytic mutations within the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) directly contributes to oncogenesis. Nevertheless, it is non-trivial to rationalize how the other high-frequency but non-catalytic mutations within the BAP1-UCH lead to malignancies. Here we used multiplex spectroscopic, thermodynamic and biophysical analyses to investigate the impacts of eleven high-occurrence mutations within BAP1-UCH on the structure, folding and function. Several mutations significantly destabilize BAP1-UCH and increase its aggregation propensity. Hydrogen-deuterium exchange mass spectrometry data revealed allosteric destabilizations caused by mutations distant from the catalytic site. Our findings gave a comprehensive and multiscale account of the molecular basis of how these non-catalytic mutations within BAP1-UCH may be implicated in oncogenesis.

Oxidation of catalytic cysteine of human deubiquitinase BAP1 triggers misfolding and aggregation in addition to functional loss.

Deubiquitinating enzymes (DUBs) form a large protease family involved in a myriad of biological and pathological processes, including ROS sensors. ROS-mediated inhibition of their DUB activities is critical for fine-tuning the stress-activated signaling pathways. Here, we demonstrate that the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) is highly sensitive to moderate oxidative stress. Oxidation of the catalytic C91 significantly destabilizes BAP1-UCH and increases the population of partially unfolded form, which is prone to aggregation. Unlike other DUBs, the oxidation-induced structural and functional loss of BAP1-UCH cannot be fully reversed by reducing agents. The oligomerization of oxidized BAP1-UCH is attributed to inter-molecular disulfide bond formation. Hydrogen-deuterium mass exchange spectrometry (HDX-MS) reveals increased fluctuations of the central ß-sheet upon oxidation. Our findings suggest that oxidation-mediated functional loss and increased aggregation propensity may contribute to oncogenesis associated with BAP1.

Converging experimental and computational views of the knotting mechanism of a small knotted protein.

MJ0366 from Methanocaldococcus jannaschii is the smallest topologically knotted protein known to date. 92 residues in length, MJ0366 ties a trefoil (31) knot by threading its C-terminal helix through a buttonhole formed by the remainder of the secondary structure elements. By generating a library of point mutations at positions pertinent to the knot formation, we systematically evaluated the contributions of individual residues to the folding stability and kinetics of MJ0366. The experimental Φ-values were used as restraints to computationally generate an ensemble of conformations that correspond to the transition state of MJ0366, which revealed several nonnative contacts. The importance of these nonnative contacts in stabilizing the transition state of MJ0366 was confirmed by a second round of mutagenesis, which also established the pivotal role of F15 in stapling the network of hydrophobic interactions around the threading C-terminal helix. Our converging experimental and computational results show that, despite the small size, the transition state of MJ0366 is formed at a very late stage of the folding reaction coordinate, following a polarized pathway. Eventually, the formation of extensive native contacts, as well as a number of nonnative ones, leads to the threading of the C-terminal helix that defines the topological knot.

Cross-over Loop Cysteine C152 Acts as an Antioxidant to Maintain the Folding Stability and Deubiquitinase Activity of UCH-L1 Under Oxidative Stress.

Redox-dependent inactivation of deubiquitinases (DUBs) is a critical factor for attenuating their DUB activity in response to cellular oxidative stress. Ubiquitin C-terminal hydrolase isoform (UCH-L1) is an important DUB that is highly expressed in human neuronal cells and is implicated in a myriad of human diseases such as neurodegenerative diseases and cancer. Increasing evidence suggests an important role of UCH-L1 in redox regulation and the protection of neuronal cells from oxidative stress. In this study, we examined the molecular basis of how UCH-L1 responds to oxidation in a reversible manner. Using H2O2 as a model oxidant, we showed by mass spectrometry that a subset of methionine and cysteine residues, namely (M1, M6, M12, C90, and C152) were more susceptible to oxidation. Spectroscopic analysis showed that oxidation of C90 can lead to profound structural changes in addition to the loss of function. Importantly, we further demonstrated that C152, which is located at the substrate recognition cross-over loop, serves as a reactive oxygen species (ROS) scavenger to protect catalytic C90 from oxidation under moderate oxidative conditions. Hydrogen-deuterium exchange mass spectrometry analysis provided detailed structural mapping of the destabilizing effect of H2O2-mediated oxidation, which resulted in global destabilization far beyond the oxidation sites. These perturbations may be responsible for irreversible aggregation when subject to prolonged oxidative stress.

Improvement of structural stability and functional efficiency of chaperonin GroEL mediated by mixed salt.

GroEL is the most commonly used chaperonin protein for both in-vitro refolding of aggregating proteins as well as in-vivo solubilization of over-expressed aggregation-prone proteins of therapeutic and biotechnological applications. But sometimes the stress conditions like heat and a load of over-expressed/unfolded/misfolded proteins lead to a decrease in structural stability and functional efficiency of GroEL, which results in less recovery of substrate protein through the chaperone-mediated refolding process. So, to amend it, we have been able to optimize physicochemical conditions utilizing a cumulation of (NH4)2SO4/MgCl2 in the buffer. Interestingly, we found a consequential enhancement in the aggregation prevention efficiency, refolding of the denatured substrate and ATPase activity of GroEL protein. The reason for the increased refolding and aggregation prevention efficiency might be the exposure of hydrophobic sites and enhanced ATP hydrolysis rate in presence of buffer containing (NH4)2SO4/MgCl2. The present study withal shows that GroEL under optimized conditions exhibits consequential amelioration in thermal aggregation at high temperature. Hence the optimized buffer conditions are utilizable for the folding of substrate proteins under a broad temperature range.

Inter and intra-subunit interactions at the subunit interface of chaperonin GroEL are essential for its stability and assembly.

Chaperonin GroEL helps in the folding of substrate proteins under normal and stress conditions. Although it remains stable and functional during stress conditions, the quantitative estimation of stability parameters and the specific amino-acid residues playing a role in its stability are not known in sufficient detail. The reason for poor understanding is its large size, multimeric nature, and irreversible unfolding process. The X-ray crystal structure reveals that equatorial domain forms almost all intra and inter-subunit interactions for assembly of GroEL. Considering all these facts, we adopted alternate strategies to use monomeric GroEL, native GroEL and equatorial domain mutants (GroELK4E/GroELD523K/GroELD473C) to study the assembly and stability of GroEL. Loss of inter-subunit interaction involving K4 residue of one subunit and E59, I60, E61, I62 residues of adjacent subunit due to K4E mutation affect the oligomerization efficiency of GroEL subunits while the equilibrium unfolding studies on wild-type monomeric GroEL, native GroEL, and the selected mutants together demonstrate that intra-subunit interactions involving K4 and D523 of the same subunit play a critical role in the thermodynamic stability of both native and monomeric GroEL without affecting the oligomerization of subunits. The stability order between the GroELwild-type(M) and its variants is GroELwild-type(M) ≥ GroELD473C(M)˃GroELD523K(M)˃GroELK4E.

Folding and unfolding pathway of chaperonin GroEL monomer and elucidation of thermodynamic parameters.

The conformation and thermodynamic stability of monomeric GroEL were studied by CD and fluorescence spectroscopy. GroEL denaturation with urea and dilution in buffer leads to formation of a folded GroEL monomer. The monomeric nature of this protein was verified by size-exclusion chromatography and native PAGE. It has a well-defined secondary and tertiary structure, folding activity (prevention of aggregation) for substrate protein and is resistant to proteolysis. Being a properly folded and reversibly refoldable, monomeric GroEL is amenable for the study of thermodynamic stability by unfolding transition methods. We present the equilibrium unfolding of monomeric GroEL as studied by urea and heat mediated unfolding processes. The urea mediated unfolding shows two transitions and a single transition in the heat mediated unfolding process. In the case of thermal unfolding, some residual structure unfolds at a higher temperature (70-75°C). The process of folding/unfolding is reversible in both cases. Analysis of folding/unfolding data provides a measure of ΔGNUH2O, Tm, ΔHvan and ΔSvan of monomeric GroEL. The thermodynamic stability parameter ΔGNUH2O is similar with both CD and intrinsic fluorescence i.e. 7.10±1.0kcal/mol. The calculated Tm, ΔHvan and ΔSvan from the thermal unfolding transition is 46±0.5°C, 43.3±0.1kcal/mol and 143.9±0.1cal/mol/k respectively.