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1. Identification of preferred multimodal ligand‐binding regions on IgG1 F C using nuclear magnetic resonance and molecular dynamics simulations

   https://doi.org/10.1002/bit.27611  

   Gudhka, Ronak B. ; Bilodeau, Camille L. ; McCallum, Scott A. ; McCoy, Mark A. ; Roush, David J. ; Snyder, Mark A. ; Cramer, Steven M. ( November 2020 , Biotechnology and Bioengineering)

   In this study, the binding of multimodal chromatographic ligands to the IgG1 F_Cdomain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated¹⁵N‐labeled F_Cdomain indicated that while single‐mode ion exchange ligands interacted very weakly throughout the F_Csurface, multimodal ligands containing negatively charged and aromatic moieties interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the F_C. The multimodal ligand‐binding sites on the F_Cwere concentrated in the hinge region and near the interface of the C_H2 and C_H3 domains. Furthermore, the multimodal binding sites were primarily composed of positively charged, polar, and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on F_Ccorroborated molecular‐level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand‐F_Cbinding in these preferred regions was shown to be electrostatic interactions and π–π stacking of surface‐exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular‐level understanding of multimodal ligand–F_Cinteractions and sets the stage for future analyses of even more complex biotherapeutics.

    

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2. Compliant mechanical response of the ultrafast folding protein EnHD under force

   https://doi.org/10.1038/s42005-022-01125-5  

   Reifs, Antonio ; Ruiz Ortiz, Irene ; Saa, Amaia Ochandorena ; Schönfelder, Jörg ; De Sancho, David ; Muñoz, Victor ; Perez-Jimenez, Raul ( December 2023 , Communications Physics)

   Abstract Ultrafast folding proteins have become an important paradigm in the study of protein folding dynamics. Due to their low energetic barriers and fast kinetics, they are amenable for study by both experiment and simulation. However, single molecule force spectroscopy experiments on these systems are challenging as these proteins do not provide the mechanical fingerprints characteristic of more mechanically stable proteins, which makes it difficult to extract information about the folding dynamics of the molecule. Here, we investigate the unfolding of the ultrafast protein Engrailed Homeodomain (EnHD) by single-molecule atomic force microscopy experiments. Constant speed experiments on EnHD result in featureless transitions typical of compliant proteins. However, in the force-ramp mode we recover sigmoidal curves that we interpret as a very compliant protein that folds and unfolds many times over a marginal barrier. This is supported by a simple theoretical model and coarse-grained molecular simulations. Our results show the ability of force to modulate the unfolding dynamics of ultrafast folding proteins. 

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3. Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc

   https://doi.org/10.3390/ijms22010055  

   Ding, Yue ; Apostolidou, Dimitra ; Marszalek, Piotr ( January 2021 , International Journal of Molecular Sciences)

   null (Ed.)

   NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies. 

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4. Biomechanical characterization of TIM protein–mediated Ebola virus–host cell adhesion

   https://doi.org/10.1038/s41598-018-36449-2  

   Dragovich, Matthew A. ; Fortoul, Nicole ; Jagota, Anand ; Zhang, Wei ; Schutt, Krista ; Xu, Yan ; Sanabria, Michelle ; Moyer, Jr., Dennis M. ; Moller-Tank, Sven ; Maury, Wendy ; et al ( January 2019 , Scientific Reports)

   Since the most recent outbreak, the Ebola virus (EBOV) epidemic remains one of the world’s public health and safety concerns. EBOV is a negative-sense RNA virus that can infect humans and non-human primates, and causes hemorrhagic fever. It has been proposed that the T-cell immunoglobulin and mucin domain (TIM) family proteins act as cell surface receptors for EBOV, and that the interaction between TIM and phosphatidylserine (PS) on the surface of EBOV mediates the EBOV–host cell attachment. Despite these initial findings, the biophysical properties of the TIM-EBOV interaction, such as the mechanical strength of the TIM-PS bond that allows the virus-cell interaction to resist external mechanical perturbations, have not yet been characterized. This study utilizes single-molecule force spectroscopy to quantify the specific interaction forces between TIM-1 or TIM-4 and the following binding partners: PS, EBOV virus-like particle, and EBOV glycoprotein/vesicular stomatitis virus pseudovirion. Depending on the loading rates, the unbinding forces between TIM and ligands ranged from 40 to 100 pN, suggesting that TIM-EBOV interactions are mechanically comparable to previously reported adhesion molecule–ligand interactions. The TIM-4–PS interaction is more resistant to mechanical force than the TIM-1–PS interaction. We have developed a simple model for virus–host cell interaction that is driven by its adhesion to cell surface receptors and resisted by membrane bending (or tension). Our model identifies critical dimensionless parameters representing the ratio of deformation and adhesion energies, showing how single-molecule adhesion measurements relate quantitatively to the mechanics of virus adhesion to the cell.

    

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5. Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy

   https://doi.org/10.1073/pnas.2020083118  

   Jacobson, David R. ; Perkins, Thomas T. ( March 2021 , Proceedings of the National Academy of Sciences)

   Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR’s G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule–derived ΔΔGfor mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔGfor mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val²¹⁷and a natively bound squalene lipid, highlighting the contribution of membrane protein–lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔGfor a fully folded membrane protein embedded in its native bilayer.

    

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