Biology subjects

Badilla, C. L.

Publications and source records attributed to Badilla, C. L..

3 recordsLinked to original sources

bioRxiv2021

Identical Sequences, Different Behaviors: ProteinDiversity Captured at the Single-Molecule Level

The classical "one sequence, one structure, one function" paradigm has shaped much of our intuition of how proteins work inside the cell. Partially due to the insight provided by bulk biochemical assays, individual biomolecules are assumed to behave as identical entities, and their characterization relies on ensemble averages that flatten any conformational diversity into a unique phenotype. While the emergence of single-molecule techniques opened the gates to interrogating individual molecules, technical shortcomings typically limit the duration of these measurements to a few minutes, which prevents to completely characterize a protein individual and, hence, to capture the heterogeneity among molecular populations. Here, we introduce a magnetic tweezers design, which showcases enhanced stability and resolution that allows us to measure the folding dynamics of a single protein during several uninterrupted days with a high temporal and spatial resolution. Thanks to this instrumental development, we do a complete characterization of two proteins with a very different force-response: the talin R3IVVI domain and protein L. Days-long recordings on the same single molecule accumulate several thousands of folding transitions sampled with sub-ms resolution, which allows us to reconstruct their free energy landscapes and describe how they evolve with force. By mapping the nanomechanical identity of many different protein individuals, we directly capture their molecular diversity as a quantifiable dispersion on their force response and folding kinetics. Our instrumental development offers a new tool for profiling individual molecules, opening the gates to the characterization of biomolecular heterogeneity.

biophysics↗

bioRxiv2021

Magnetic tweezers meets AFM: ultra-stable protein dynamics across the force spectrum

Proteins that operate under force--cell adhesion, mechanosensing--exhibit a wide range of mechanostabilities. Single-molecule magnetic tweezers has enabled the exploration of the dynamics under force of these proteins with subpiconewton resolution and unbeatable stability in the 0.1-120 pN range. However, proteins featuring a high mechanostability (>120 pN) have remained elusive with this technique and have been addressed with Atomic Force Microscopy (AFM), which can reach higher forces but displays less stability and resolution. Herein, we develop a magnetic tweezers approach that can apply AFM-like mechanical loads while maintaining its hallmark resolution and stability in a range of forces that spans from 1 to 500 pN. We demonstrate our approach by exploring the folding and unfolding dynamics of the highly mechanostable adhesive protein FimA from the Gram-positive pathogen Actinomyces oris. FimA unfolds at loads >300 pN, while its folding occurs at forces <15 pN, producing a large dissipation of energy that could be crucial for the shock absorption of mechanical challenges during host invasion. Our novel magnetic tweezers approach entails an all-in-one force spectroscopy technique for protein dynamics studies across a broad spectrum of physiologically-relevant forces and timescales.

biophysics↗

bioRxiv2018

Isopeptide-Blocker Impairs the Mechanics of Recently Translated Pilin Proteins

Bacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of Gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen Streptococcus pyogenes, resulting in mechanically labile pilin domains. We use a combination of protein engineering and AFM force spectroscopy to demonstrate that the peptide blocks the formation of the native isopeptide bond and compromises the mechanics of the domain. While an intact Spy0128 is inextensible at any force, peptide-modified Spy0128 pilins readily unfold at very low forces, marking the abrogation of the intramolecular isopeptide bond as well as the absence of a stable pilin fold. We propose that isopeptide-blocking peptides could be further developed as a novel type of highly-specific anti-adhesive antibiotics to treat Gram-positive pathogens.\n\nSignificanceAt the onset of an infection, Gram-positive bacteria adhere to host cells through their pili, filamentous structures built by hundreds of repeats of pilin proteins. These proteins can withstand large mechanical challenges without unfolding, remaining anchored to the host and resisting cleavage by proteases and oxygen radicals present in the targeted tissues. The key structural component that gives pilins mechanical resilience are internal isopeptide bonds, strategically placed so that pilins become inextensible structures. We target this bond by designing a blocking peptide that interferes with its formation during folding. We demonstrate that peptide-modified pilins lack mechanical stability and extend at low forces. We propose this strategy as a rational design of mechanical antibiotics, targeting the Achilles Heel of bacterial adhesion.

biophysics↗