1. Mechanical biosystems: Deciphering mechanical properties of biomolecules
Keywords: Molecular biomechanics, atomic force microscopy, single-molecule force spectroscopy, single-molecule biophysics
Proteins, essential molecular machines within cells, are routinely influenced by mechanical forces that modulate their function. These forces are integral to numerous biological processes, such as muscle contraction, blood coagulation, auditory perception, cell adhesion, and even bacterial pathogenesis1. Additionally, the effect of molecular forces on engineered protein therapeutics is an evolving area of study.
The Nash Lab focuses on understanding these mechanical influences at a molecular level. We utilize the atomic force microscope (AFM) and high-throughput shear flow assays2 to measure the force resistance of molecules and molecular complexes.
Through single-molecule force spectroscopy (SMFS), we quantify the energy landscapes that dictate protein folding/unfolding and receptor-ligand unbinding dynamics under applied force3-5. We analyze and engineer protein-protein interactions that strengthen when force is applied (i.e. catch bonds), which are analogous to a molecular finger trap, and study how anchor point chemistry can influence the deformation and unbinding of biomolecules6,7. By harnessing the capabilities of single-molecule techniques, our aim is to derive a comprehensive understanding of the mechanics underlying protein interactions. This knowledge will be pivotal for the strategic design and engineering of biological systems in the future.
- Liu, H., et al. JACS Au, 2(6): 1417-1427, (2022); doi:10.1021/jacsau.2c00121
- Santos, M. S., et al. Biophysical Reports, 2(1), 10035, (2022); doi:10.1016/j.bpr.2021.100035
- Yang, B., et al. Nano letters 20.12 (2020): 8940-8950; doi: 10.1021/acs.nanolett.0c04178
- Liu, Z., et al. Nature Communications 11, 4321 (2020); doi: 10.1038/s41467-020-18063-x
- Liu, H., et al. Nano Letters, 19(8): 5524-5529, (2019); doi: 10.1021/acs.nanolett.9b02062
- Liu, Z., et al. Nano Letters 22(1): 179–187, (2021); doi: 10.1021/acs.nanolett.1c03584
- Liu, H., et al. Angew. Chem. Int. Ed. (2023); doi: 10.1002/anie.202304136
2. Biomolecular engineering: Directed evolution, high-throughput screening and DNA sequencing
Keywords: High-throughput protein engineering, next-generation sequencing, machine learning, laboratory directed evolution, biologics, therapeutics, diagnostics
The pharmaceutical industry is rapidly transitioning from small molecule drugs to biologics. However, naturally occurring proteins frequently lack the requisite stability, specificity, developability, and function to be directly deployed as biopharmaceuticals. In order to enhance the molecular properties of therapeutic proteins, the Nash Lab develops novel methods to analyse sequence-structure-function relationships in a high-throughput manner. We develop screening techniques tailored to specific molecular functions that preserve genotype-phenotype linkage 1–3. These methods are integrated with single cell sorting and next-generation sequencing (NGS) in ultra-high-throughput (HT) workflows, allowing us to investigate the impact of thousands of mutations on protein fitness4. Using the acquired insights, we construct custom combinatorial mutant libraries to be employed in directed evolution (DE) experiments, ultimately leading to the identification of improved protein variants5,6. Additionally, we harness computational tools and machine learning (ML) algorithms to aid the design of mutant libraries and conduct in-silico screening over an extensive combinatorial space7. Our ultimate objective is to enhance protein therapeutics and gain a deeper understanding of the structural and functional mechanisms inherent in natural proteins. This knowledge will significantly streamline protein engineering workflows, facilitating the successful development of biologics for clinical applications.
- Vanella, R. et al. Chem. Mater. (2019) doi: 10.1021/acs.chemmater.8b04348
- Lopez-Morales, J. et al. ACS Synth. Biol. 12, 419–431 (2023)
- Fernández De Santaella J. et al. Anal. Chem. 95, 7150–7157 (2023)
- Vanella, R. et al. BioRxiv 2023.02.24.529916 (2023) doi: 10.1101/2023.02.24.529916
- Vanella, R. et al. Biotechnol. Bioeng. (2019) doi: 10.1002/bit.27002
- Vanella, R. et al. Chem. Commun. 58, 2455–2467 (2022).
3. Molecular sensors: Stimuli-responsive protein-based polymers
Keywords: Smart polymers, environmentally responsive proteins, elastin-like polypeptides
Stimuli-responsive or 'smart' polymers undergo dramatic conformational changes in response to slight variations in environmental conditions. Examples include small changes in pH, temperature, salt, and light that give rise to collapse or expansion of polymer chains in aqueous media. We work with both synthetic polymers and elastin-like polypeptides (repetitive protein polymers) that possess 'smart' properties. Our focus is on integrating smart materials into physiological processes to modulate biomolecular activity and related physiological processes in a predictable manner. For instance, in the coagulation cascade, by introducing recognition sites of FXIIIa, we managed to modulate the process of fibrinogen assembly in a temperature-responsive manner, thereby modulating the structure of the fibrin crosslinking network and improving biomechanical properties of blood clots. This advancement holds great promise for addressing severe pathological bleeding and coagulation disorders.
- Nash, Michael A. "Elastin-like Polypeptides: Protein-based Polymers for Biopharmaceutical Development: Medicinal Chemistry and Chemical Biology Highlights." Chimia 76.5 (2022): 478-478.
- Risser, Fanny, Joanan López-Morales, and Michael A. Nash. "Adhesive Virulence Factors of Staphylococcus aureus Resist Digestion by Coagulation Proteases Thrombin and Plasmin." ACS bio & med Chem Au 2.6 (2022): 586-599.
- Risser, Fanny, et al. "Engineered molecular therapeutics targeting fibrin and the coagulation system: a biophysical perspective." Biophysical reviews 14.2 (2022): 427-461.
- Urosev, Ivan, Joanan Lopez Morales, and Michael A. Nash. "Phase separation of intrinsically disordered protein polymers mechanically stiffens fibrin clots." Advanced Functional Materials 30.51 (2020): 2005245.