Biology subjects

Ramirez-Sarmiento, C. A.

Publications and source records attributed to Ramirez-Sarmiento, C. A..

9 recordsLinked to original sources

bioRxiv2026

Exploration of the structural and functional diversity in the metamorphic RfaH subfamily

RfaH, a member of the universally conserved NusG/Spt5 family, activates transcription and translation of virulence genes by bridging the transcribing RNA polymerase to the translating ribosome. Unlike its monomorphic, constitutively active paralog NusG, RfaH fold-switches from an autoinhibited state, wherein its C-terminal domain forms an -helical hairpin bound to the N-terminal domain, into an active NusG-like state through C-terminal domain dissociation and refolding into a {beta}-barrel. RfaH has been proposed to evolve from NusG via gene duplication by first activating genes adjacent to its production site, before developing autoinhibition to selectively control distant genes. Using AlphaFold2, we predicted the structures of thousands of RfaH homologs and identified a subset ([~]14%) that appear predominantly folded in the active state. In vivo assays confirmed that these putative monomorphic homologs exhibit constitutive activity comparable to known RfaH mutants. Phylogenetic and genomic analyses revealed that these monomorphic proteins form a distinct clade and are preferentially located within or next to long virulence-associated operons. Together, these results further support a stepwise evolutionary model of RfaH specialization through structural transformation.

biochemistry↗

bioRxiv2026

MPNN-guided redesign of PET hydrolases with enhanced catalytic activity below the PET glass transition temperature

Enzymatic depolymerization of polyethylene terephthalate (PET) presents a sustainable route for plastic circularity, but its industrial viability is disadvantaged by the need for thermostable enzymes active under mild, energy-efficient conditions. While Polyester Hydrolase Leipzig 7 (PHL7, also known as PES-H1) rapidly degrades amorphous PET near the glass transition temperature of this polymer (~65{degrees}C), its poor protein expression, inactivation above 60{degrees}C and slow depolymerization below 60{degrees}C limits its practical application. Here, we employ ProteinMPNN and LigandMPNN, structural and evolutionary information, to redesign the sequence of PHL7 and improve protein expression, thermostability and activity. We identified 2/36 experimentally tested variants (D5, D11) with enhanced PET depolymerization at 50{degrees}C, achieving the same efficiency as PHL7 at 70{degrees}C but with a shifted product profile, favoring mono-(2-hydroxyethyl) terephthalate (MHET) over terephthalate. Molecular dynamics revealed that these redesigns exhibit enhanced flexibility in active site regions, providing a mechanistic understanding of their low-temperature catalysis. These variants enable a potential route to resynthesize virgin PET via MHET polycondensation, offering an efficient circular economy pathway.

bioengineering↗

bioRxiv2026

Polar Marine Microbial Communities as Reservoirs of Polyester Degrading Enzymes

BackgroundPolyethylene terephthalate (PET) is one of the most widely used plastics and a major contributor to marine pollution. While the diversity of PET hydrolases (PETases), which degrade PET into mono(2-hydroxyethyl) terephthalate (MHET), terephthalate (TPA) and ethylene glycol, has been documented in temperate and tropical waters, their potential presence in polar oceans remain unascertained. ResultsHere, we systematically screened polar and non-polar marine metagenomes using Hidden Markov models (HMM) generated using experimentally validated PETases. We identified >680 putative PETase-like sequences, with Antarctic and Arctic candidates enriched in high-fidelity motifs associated with PETase-like activity. Phylogenetic and structural analyses defined a high-confidence PETase-like clade comprising both Type I and Type II enzymes, differing in thermostability-related features and PET-binding motifs. Experimental assays confirmed polyesterase activity in 5/9 candidates from this clade, including polar-derived variants active at 14-25{degrees}C. Downstream enzymes for PET consumption were also widespread, detecting 209 putative MHET hydrolases and 442 TPA-catabolyzing enzymes. Further, we reconstructed 112 metagenome-assembled genomes (MAGs) carrying at least one PETase-like gene, more than half from polar datasets. Notably, 15 MAGs encoded multiple PETase-like enzymes, and 1 Antarctic MAG harbored a complete PETase-MHETase-TPA pathway, evidencing a fully integrated degradation potential in cold-adapted taxa. ConclusionsTogether, these results demonstrate that polar oceans act as previously overlooked reservoirs of taxonomically and functionally diverse plastic-degrading enzymes. The enrichment of PETase-like enzymes and downstream pathways in polar microbial communities expands the global biogeography of plastic biodegradation and highlights cold-active enzymes as promising candidates for developing low-temperature plastic bioremediation strategies.

bioinformatics↗

bioRxiv2026

FrustraMPNN: An ultra-fast deep learning tool for proteome-scale analysis of deep mutational single-residue local energetic frustration in proteins

Energetic frustration, characterized by conflicting local interactions, is a key determinant of protein dynamics, allostery, protein-protein interactions, enzyme catalysis, and overall protein function. Although the principle of minimal frustration describes an evolutionary bias towards a reduction of energetic conflicts for efficient protein folding, local violations are selectively embedded to encode the complex energy landscapes necessary for protein functions. However, the computational cost of traditional single-residue frustration analysis has prevented the calculation of these energetic conflicts at the proteome scale, creating a significant bottleneck in structural biology. Here, we introduce FrustraMPNN, a message-passing neural network retrained via transfer learning that predicts a complete per-residue frustration-index mutation profile (full mutational scanning per position for a whole protein) orders-of-magnitude faster than existing methods while maintaining high accuracy. This is demonstrated by calculating the single-residue frustration of the E. coli proteome, reducing the calculation time from years to 12 hours. We validate FrustraMPNN on a diverse external set of over 3,400 human protein structures, achieving a Spearman correlation of up to 0.80 across all frustration categories, demonstrating robust generalizability. By conducting a thorough dataset ablation study, we find that the models performance improves when trained on a broader range of protein sizes, highlighting an inherent limitation of datasets like Megascale. We provide FrustraMPNN as an open-source tool, expecting it will enable exploration of new hypotheses in fields such as personalized structural biology and allow analysis of local energetic frustration patterns involved in protein function at an unprecedented proteomic scale.

bioinformatics↗

bioRxiv2025

Structural and Biophysical Basis for PFAS Binding by Human Sterol Carrier Protein-2

Per- and polyfluoroalkyl substances (PFAS) are harmful environmental contaminants that bioaccumulate in human tissues and are linked to adverse health outcomes. While PFAS are known to bind to a variety lipid binding proteins (LBPs), such as human serum albumin and fatty acid-binding proteins (FABPs), the broader molecular basis for their biological distribution and additional target proteins in humans remains unanswered. Motivated by its known promiscuity towards a range of hydrophobic ligands, we investigated the interaction between human sterol carrier protein 2 (SCP2) and various PFAS. SCP2 is a structurally distinct LBP with no previously reported affinity for PFAS. Using a combination of screening, fluorescence displacement assays, protein structure prediction of PFAS-SCP2 complexes, ITC, and NMR experiments, we demonstrate for the first time that SCP2 is a PFAS-binding protein. Our findings provide insight into the residues participating in these interactions and provide evidence for an additional LBP that may facilitate PFAS distribution and persistence in the human body.

biophysics↗

bioRxiv2024

Open Educational Resources for distributed hands-on teaching in molecular biology

The urgent need to develop a more equitable bioeconomy has positioned biotechnology capacity building at the forefront of international priorities. However, in many educational institutions, particularly in low-and middle-income countries, this remains a major challenge due to limited access to reagents, equipment, and technical documentation. In this work, we describe Open Educational Resources (OER) composed of locally produced biological reagents, open source hardware and free software to teach fundamental techniques in biotechnology such as LAMP DNA amplification, RT-PCR RNA detection, enzyme kinetics and fluorescence imaging. The use of locally produced reagents and devices reduces costs by up to one order of magnitude. During the pandemic lockdowns, these tools were distributed nationwide to students homes as a lab-in-a-box for remote teaching of molecular biology. To test their performance, a total of 93 undergraduate students tested these resources during a biochemistry practical course. 27 out of 31 groups ([~]87%) successfully achieved the objectives of the PCR activity, while 28 out of 31 groups ([~]90%) correctly identified the target using LAMP reactions. To assess the potential application in secondary school, we organized three workshops for high school teachers from different institutions across Chile and performed an anonymous questionnaire, obtaining a strong agreement on how these OER broaden teachers perspectives on the techniques and facilitate the teaching of molecular biology topics. This effort was made possible through a close collaboration with open source technology advocates and members of DIYbio communities, whose work is paving the way for low-cost training in biology. All the protocols and design files are available under open source licenses.

molecular biology↗

bioRxiv2023

Concerted transformation of a hyper-paused transcription complex and its reinforcing protein

RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an -helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a {beta}-barrel, which binds the ribosome. Our structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH reveal swiveled, pre-translocated pause states stabilized by an ops hairpin in the non-template DNA. Autoinhibited RfaH binds and twists the ops hairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by the ops and RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life. HIGHLIGHTS- The nontemplate DNA strand of an ops-paused transcription complex forms a hairpin - Autoinhibited RfaH binds and twists the ops hairpin to expand the RNA:DNA hybrid - RfaH-hairpin contacts are solely responsible for triggering RfaH activation - Upon recruitment, RfaH hyper-stabilizes the pause and promotes RNAP backtracking

biochemistry↗

bioRxiv2021

The N-terminal domain of RfaH plays an active role in protein fold-switching

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) stalled at a DNA signal known as ops. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a {beta}-barrel while RfaH CTD is forming an -hairpin blocking such site. Upon recognition of the ops exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a {beta}-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, most studies employ tertiary biases towards each native state, hampering the analysis of sequence-encoded interactions on fold-switching. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards -folded CTD, whereas the NTD interferes with {beta}CTD folding, becoming trapped in a {beta}-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the -folded RfaH by specifically orienting the CTD upon interdomain binding and also by favoring {beta}-barrel rupture into an intermediate from which fold-switching proceeds.

biophysics↗

bioRxiv2019

Differential local stability governs the metamorphic fold-switch of bacterial virulence factor RfaH

A regulatory factor RfaH, present in many Gram-negative bacterial pathogens, is required for transcription and translation of long operons encoding virulence determinants. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA sequence within these operons. Upon recruitment, the C-terminal domain of this two-domain protein refolds from an -hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound {beta}-barrel that interacts with the ribosome to enable translation. Although structures of the autoinhibited (-hairpin) and active ({beta}-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125-145) is stably folded in the autoinhibited state ([~]20% deuteron incorporation), while the rest of this domain is highly flexible (>40% deuteron incorporation) and its flexibility only decreases in the {beta}-folded state. Computationally-predicted {Delta}Gs agree with these results by displaying similar anisotropic stability within the tip of the -hairpin and on neighboring N-terminal domain residues. Remarkably, the {beta}-folded state shows comparable stability to non-metamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins, and for devising targeted strategies to combat bacterial diseases.\n\nSignificanceInfections caused by Gram-negative bacteria are a worldwide health threat due to rapid acquisition of antibiotic resistance. RfaH, a protein essential for virulence in several Gram-negative pathogens, undergoes a large-scale structural rearrangement in which one RfaH domain completely refolds. Refolding transforms RfaH from an inactive state that restricts RfaH recruitment to a few target genes into an active state that binds to, and couples, transcription and translation machineries to elicit dramatic activation of gene expression. However, the molecular basis of this unique conformational change is poorly understood. Here, we combine molecular dynamics and structural biology to unveil the hotspots that differentially stabilize both states of RfaH. Our findings provide novel insights that will guide design of inhibitors blocking RfaH action.

biophysics↗