A method to measure nanomechanical properties of biological objects

We postulate that one will be able to quantitatively infer changes in the mechanical properties of proteins, cells, and other biological objects (BO) by measuring the shifts of several thermally excited resonance frequencies of atomic force microscopy cantilevers in contact with BOs. Here, we provide a method to extract spring constants and molecular damping factors of BOs in biologically relevant phosphate buffered saline medium and using compliant AFM cantilevers with a small aspect ratio (a ratio of length to width)

Nanomechanical properties of single protein molecules and peptides

Master of ScienceDepartment of PhysicsRobert SzoszkiewiczProteins are involved in many of the essential cellular processes, such as cell adhesion, muscle function, enzymatic activity or signaling. It has been observed that the biological function of many proteins is critically connected to their folded conformation. Thus, the studies of the process of protein folding have become one of the central questions at the intersection of biophysics and biochemistry. We propose to use the changes of the nanomechanical properties of these biomolecules as a proxy to study how the single proteins fold. In the first steps towards this goal, the work presented in this thesis is concentrated on studies of unfolding forces and pathways of one particular multidomain protein, as well as on development of the novel method to study elastic spring constant and mechanical energy dissipation factors of simple proteins and peptides. In the first part of this thesis we present the results of the mean unfolding forces of the NRR region of the Notch1 protein. Those results are obtained using force spectroscopy techniques with the atomic force microscope (AFM) on a single molecule level. We study force-induced protein unfolding patterns and relate those to the conformational transitions within the protein using available crystal structure of the Notch protein and molecular dynamics simulations. Notch is an important protein, involved in triggering leukemia and breast cancers in metazoans, i.e., animals and humans. In the second part of this thesis we develop a model to obtain quantitative measurements of the molecular stiffness and mechanical energy dissipation factors for selected simple proteins and polypeptides from the AFM force spectroscopy measurements. We have developed this model by measuring the shifts of several thermally excited resonance frequencies of atomic force microscopy cantilevers in contact with the biomolecules. Next, we provided partial experimental validation of this model using peptide films. Ultimately, our results are expected to contribute in the future to the developments of medical sciences, which are advancing at a level, where human health and disease can be traced down to molecular scale

Improving sampling of crystallographic disorder in ensemble refinement

Ensemble refinement, the application of molecular dynamics to crystallographic refinement, explicitly models the disorder inherent in macromolecular structures. These ensemble models have been shown to produce more accurate structures than traditional single-model structures. However, suboptimal sampling of the molecular-dynamics simulation and modelling of crystallographic disorder has limited the utility of the method, and can lead to unphysical and strained models. Here, two improvements to the ensemble refinement method implemented within Phenix are presented: DEN restraints, which guide the local sampling of conformations and allow a more robust exploration of local conformational landscapes, and ECHT disorder models, which allow the selection of more physically meaningful and effective disorder models for parameterizing the continuous disorder components within a crystal. These improvements lead to more consistent and physically interpretable simulations of macromolecules in crystals, and allow structural heterogeneity and disorder to be systematically explored on different scales. The new approach is demonstrated on several case studies and the SARS-CoV-2 main protease, and demonstrates how the choice of disorder model affects the type of disorder that is sampled by the restrained molecular-dynamics simulation

Novel staphylococcal inhibitors of neutrophil granule enzymes

Doctor of PhilosophyDepartment of Biochemistry and Molecular BiophysicsBrian V. GeisbrechtNeutrophils are our most abundant white blood cells and the first leukocytes to infiltrate sites of infection or damaged/healing tissue. Activation of neutrophils results in the mobilization of several types of granules stored within their cytosol, such as the so-called azurophilic granules, which either fuse with the maturing endophagocytic compartment or are released into the extracellular environment. One of the most abundant component of zurophilic granules is a heme-containing enzyme called myeloperoxidase (MPO), which reduces the H₂O₂ produced by the neutrophil’s respiratory burst to generate cytotoxic hypohalous acids, most typically HOCl. While neutrophil granule enzymes are essential for our innate defenses, neutrophil-driven inflammation outside this beneficial context lies at the heart of many non-infectious human diseases. Staphylococcus aureus and closely related species are highly adapted to their hosts and have evolved many strategies to resist opsonization and phagocytosis. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium can actively evade specific intracellular killing mechanisms used by neutrophils. Recent work found a highly conserved S. aureus protein, SPIN (for Staphylococcal Peroxidase INhibitor), that specifically binds and inhibits MPO [1]. This study was focused on characterizing the structure/function relationship for MPO inhibitors, SPIN proteins. To identify key residues for SPIN function in more detail, we examined two types of SPIN proteins using structural methods, direct binding assays, and functional assays for MPO activity: deletion mutants and SPIN proteins originating from divergent staphylococcal species. Together, these studies shed light on the molecular features which determine the specificity of SPIN proteins for MPO and suggest potential avenues for using this information toward the design of synthetic MPO inhibitors. In addition to the focus on targeted inhibition of MPO for its therapeutic value in treatment of a number of significant human inflammatory diseases, our investigations contributed in expanding our knowledge on infection spreading. As a first cellular host defense response, the neutrophil interaction with pathogens are of major interest. Characterization of staphylococcal immune evasion proteins is vital for understanding bacterial survival when encountering neutrophils and their bioactive constituents

Improving sampling of crystallographic disorder in ensemble refinement

Ensemble refinement, the application of molecular dynamics to crystallographic refinement, explicitly models the disorder inherent in macromolecular structures. These ensemble models have been shown to produce more accurate structures than traditional single-model structures. However, suboptimal sampling of the molecular-dynamics simulation and modelling of crystallo­graphic disorder has limited the utility of the method, and can lead to unphysical and strained models. Here, two improvements to the ensemble refinement method implemented within Phenix are presented: DEN restraints, which guide the local sampling of conformations and allow a more robust exploration of local conformational landscapes, and ECHT disorder models, which allow the selection of more physically meaningful and effective disorder models for parameterizing the continuous disorder components within a crystal. These improvements lead to more consistent and physically interpretable simulations of macromolecules in crystals, and allow structural heterogeneity and disorder to be systematically explored on different scales. The new approach is demonstrated on several case studies and the SARS-CoV-2 main protease, and demonstrates how the choice of disorder model affects the type of disorder that is sampled by the restrained molecular-dynamics simulation

Improving sampling of crystallographic disorder in ensemble refinement

Ensemble refinement, the application of molecular dynamics to crystallographic refinement, explicitly models the disorder inherent in macromolecular structures. These ensemble models have been shown to produce more accurate structures than traditional single-model structures. However, suboptimal sampling of the molecular-dynamics simulation and modelling of crystallographic disorder has limited the utility of the method, and can lead to unphysical and strained models. Here, two improvements to the ensemble refinement method implemented within Phenix are presented: DEN restraints, which guide the local sampling of conformations and allow a more robust exploration of local conformational landscapes, and ECHT disorder models, which allow the selection of more physically meaningful and effective disorder models for parameterizing the continuous disorder components within a crystal. These improvements lead to more consistent and physically interpretable simulations of macromolecules in crystals, and allow structural heterogeneity and disorder to be systematically explored on different scales. The new approach is demonstrated on several case studies and the SARS-CoV-2 main protease, and demonstrates how the choice of disorder model affects the type of disorder that is sampled by the restrained molecular-dynamics simulation

Identification and structural characterization of a novel myeloperoxidase inhibitor from Staphylococcus delphini

Staphylococcus aureus and related species are highly adapted to their hosts and have evolved numerous strategies to evade the immune system. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium evades intracellular killing mechanisms used by neutrophils. We recently discovered an S. aureus protein (SPIN for Staphylococcal Peroxidase INhibitor) that binds to and inhibits myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. To allow for comparative studies between multiple SPIN sequences, we identified a panel of homologs from species closely related to S. aureus. Characterization of these proteins revealed that SPIN molecules from S. agnetis, S. delphini, S. schleiferi, and S. intermedius all bind human MPO with nanomolar affinities, and that those from S. delphini, S. schleiferi, and S. intermedius inhibit human MPO in a dose-dependent manner. A 2.4 Å resolution co-crystal structure of SPIN-delphini bound to recombinant human MPO allowed us to identify conserved structural features of SPIN proteins, and to propose sequence-dependent physical explanations for why SPIN-aureus binds human MPO with higher affinity than SPIN-delphini. Together, these studies expand our understanding of MPO binding and inhibition by a recently identified component of the staphylococcal innate immune evasion arsenal

Single Molecule Studies of Force-Induced S2 Site Exposure in the Mammalian Notch Negative Regulatory Domain

Notch signaling in metazoans is responsible for key cellular processes related to embryonic development and tissue homeostasis. Proteolitic cleavage of the S2 site within an extracellular NRR domain of Notch is a key early event in Notch signaling. We use single molecule force–extension (FX) atomic force microscopy (AFM) to study force-induced exposure of the S2 site in the NRR domain from mouse Notch 1. Our FX AFM measurements yield a histogram of N-to-C termini lengths, which we relate to conformational transitions within the NRR domain. We detect four classes of such conformational transitions. From our steered molecular dynamics (SMD) results, we associate first three classes of such events with the S2 site exposure. AFM experiments yield their mean unfolding forces as 69 ± 42, 79 ± 45, and 90 ± 50 pN, respectively, at 400 nm/s AFM pulling speeds. These forces are matched by the SMD results recalibrated to the AFM force loading rates. Next, we provide a conditional probability analysis of the AFM data to support the hypothesis that a whole sequence of conformational transitions within those three clases is the most probable pathway for the force-induced S2 site exposure. Our results support the hypothesis that force-induced Notch activation requires ligand binding to exert mechanical force not in random but in several strokes and over a substantial period of time