Browsing by Author "Ramirez-Sarmiento, Cesar A."

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    Active Site Flexibility as a Hallmark for Efficient PET Degradation by I-sakaiensis petase
    (2018) Fecker, Tobias; Galaz Davison, Pablo; Engelberger, Felipe; Narui, Yoshie; Sotomayor, Marcos; Parra, Loreto; Ramirez-Sarmiento, Cesar A.
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    An Open One-Step RT-qPCR for SARS-CoV-2 detection
    (2024) Cerda, Ariel; Rivera, Maira; Armijo, Grace; Ibarra-Henriquez, Catalina; Reyes, Javiera; Blazquez-Sanchez, Paula; Aviles, Javiera; Arce, Anibal; Seguel, Aldo; Brown, Alexander J.; Vasquez, Yesseny; Cortez-San Martin, Marcelo; Cubillos, Francisco A.; Garcia, Patricia; Ferres, Marcela; Ramirez-Sarmiento, Cesar A.; Federici, Fernan; Gutierrez, Rodrigo A.
    The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.
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    Antarctic Polyester Hydrolases Degrade Aliphatic and Aromatic Polyesters at Moderate Temperatures
    (2022) Blazquez-Sanchez, Paula; Engelberger, Felipe; Cifuentes-Anticevic, Jeronimo; Sonnendecker, Christian; Grinen, Aransa; Reyes, Javiera; Diez, Beatriz; Guixe, Victoria; Richter, P. Konstantin; Zimmermann, Wolfgang; Ramirez-Sarmiento, Cesar A.
    Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60 degrees C and 70 degrees C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25 degrees C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modeling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases.
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    Dimer dissociation is a key energetic event in the fold-switch pathway of KaiB
    (2022) Rivera, Maira; Galaz-Davison, Pablo; Retamal-Farfan, Ignacio; Komives, Elizabeth A.; Ramirez-Sarmiento, Cesar A.
    Cyanobacteria possesses the simplest circadian clock, composed of three proteins that act as a phosphorylation oscillator: KaiA, KaiB, and KaiC. The timing of this oscillator is determined by the fold-switch of KaiB, a structural rearrangement of its C-terminal half that is accompanied by a change in the oligomerization state. During the day, KaiB forms a stable tetramer (gsKaiB), whereas it adopts a monomeric thioredoxin-like fold during the night (fsKaiB). Although the structures and functions of both native states are well studied, little is known about the sequence and structure determinants that control their structural interconversion. Here, we used confinement molecular dynamics (CCR-MD) and folding simulations using structure-based models to show that the dissociation of the gsKaiB dimer is a key energetic event for the fold-switch. Hydrogen-deuterium ex-change mass spectrometry (HDXMS) recapitulates the local stability of protein regions reported by CCR-MD, with both ap-proaches consistently indicating that the energy and backbone flexibility changes are solely associated with the region that fold-switches between gsKaiB and fsKaiB and that the localized regions that differentially stabilize gsKaiB also involve regions outside the dimer interface. Moreover, two mutants (R23C and R75C) previously reported to be relevant for altering the rhyth-micity of the Kai clock were also studied by HDXMS. Particularly, R75C populates dimeric and monomeric states with a deute-rium incorporation profile comparable to the one observed for fsKaiB, emphasizing the importance of the oligomerization state of KaiB for the fold-switch. These findings suggest that the information necessary to control the rhythmicity of the cyanobacterial biological clock is, to a great extent, encoded within the KaiB sequence.
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    Domain tethering impacts dimerization and DNA-mediated allostery in the human transcription factor FoxP1
    (2023) Cruz, Perla; Paredes, Nicolas; Asela, Isabel; Kolimi, Narendar; Molina, Jose Alejandro; Ramirez-Sarmiento, Cesar A.; Goutam, Rajen; Huang, Gangton; Medina, Exequiel; Sanabria, Hugo
    Transcription factors are multidomain proteins with specific DNA binding and regulatory domains. In the human FoxP subfamily (FoxP1, FoxP2, FoxP3, and FoxP4) of transcription factors, a 90 residue-long disordered region links a Leucine Zipper (ZIP)-known to form coiled-coil dimers-and a Forkhead (FKH) domain-known to form domain swapping dimers. We used replica exchange discrete molecular dynamics simulations, single-molecule fluorescence experiments, and other biophysical tools to understand how domain tethering in FoxP1 impacts dimerization at ZIP and FKH domains and how DNA binding allosterically regulates their dimerization. We found that domain tethering promotes FoxP1 dimerization but inhibits a FKH domain-swapped structure. Furthermore, our findings indicate that the linker mediates the mutual organization and dynamics of ZIP and FKH domains, forming closed and open states with and without interdomain contacts, thus highlighting the role of the linkers in multidomain proteins. Finally, we found that DNA allosterically promotes structural changes that decrease the dimerization propensity of FoxP1. We postulate that, upon DNA binding, the interdomain linker plays a crucial role in the gene regulatory function of FoxP1.
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    Engineering the catalytic activity of an Antarctic PET-degrading enzyme by loop exchange
    (2023) Blazquez-Sanchez, Paula; Vargas, Jhon A.; Furtado, Adriano A.; Grinen, Aransa; Leonardo, Diego A.; Sculaccio, Susana A.; Pereira, Humberto D'Muniz; Sonnendecker, Christian; Zimmermann, Wolfgang; Diez, Beatriz; Garratt, Richard C.; Ramirez-Sarmiento, Cesar A.
    Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25 degrees C to 40 degrees C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25 degrees C. First, we obtained the crystal structure of Mors1 at 1.6 A resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45 degrees C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25 degrees C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.