Biology subjects

Pardo-Avila, F.

Publications and source records attributed to Pardo-Avila, F..

6 recordsLinked to original sources

bioRxiv2026

Quasi-continuous cotranslational compaction and folding of a multidomain protein

Most proteins start to fold cotranslationally as they come off the ribosome. So far, studies of cotranslational folding have focused mainly on small, single-domain proteins. Here, we have used Force Profile Analysis to study the cotranslational folding of Firefly luciferase, a complex 550-residue protein composed of an N-terminal domain (NTD) encompassing two split Rossmann folds (RF-1, RF-2) and a {beta}-roll, and a flexibly attached C-terminal domain (CTD). The folding process is characterized by a quasi-continuous series of compaction/folding steps that generate intermediate-size pulling forces on the nascent chain, punctuated by a prominent high-force event that represents the folding of the RF-2 domain, and a few low-force instances that likely indicate the formation of distinct folding intermediates. Trigger Factor interacts extensively with the nascent chain when the central part of RF-2 and the early parts of the CTD are synthesized. Our analysis uncovers a cotranslational compaction/folding process that is rich in detail and not just a simple succession of a few distinct, cooperative folding transitions.

molecular biology↗

bioRxiv2025

PDBCleanV2: A Python Library for Generating Consistent Structure Datasets

The number of structures in the Protein Data Bank has grown rapidly in recent years. Nonetheless, comparing structures of a specific system often proves challenging due to variant nomenclature, origins from different species, presence of various ligands, and missing atoms or chains. To address these issues, we have developed PDBCleanV2, a Python package that enables users to create consistent datasets of structures, simplifying comparison among structures. This library generates individual files for each molecule in a structure file, corrects labeling errors, and standardizes chain names and numbering. Our aim is to provide researchers with consistent datasets that streamline their analysis. The source code, installation instructions, and tutorials can be found in PDBCleanV2s GitHub repository and Zenodo accessible at https://github.com/fatipardo/PDBClean-0.0.2/ and https://doi.org/10.5281/zenodo.14014241, respectively.

bioinformatics↗

bioRxiv2021

Probing interplays between human XBP1u translational arrest peptide and 80S ribosome

The ribosome stalling mechanism is a crucial biological process; yet its atomistic underpinning is still elusive. In this framework, the XBP1u translational arrest peptide (AP) plays a central role in regulating the Unfolded Protein Response (UPR) in eukaryotic cells. Here, we report multi-microseconds all atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wild type AP and for four mutant variants of different arrest potency. Enhanced sampling simulations allow investigating the AP release process of the different variants shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP-ribosome interactions.

molecular biology↗

bioRxiv2018

Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature

The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic vs ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44/h45 interface in varying states, that is, engaged, disengaged and in equilibrium. Thirdly, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44/h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

biophysics↗

bioRxiv2018

The Shape of the Ribosome Exit Tunnel Affects Cotranslational Protein Folding

The E.coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 {Delta}loop ribosomes, while two ~100-residue protein normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 {Delta}loop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

biochemistry↗

bioRxiv2018

Structure of the 30S ribosomal decoding complex at ambient temperature

The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New lightsources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 [A] resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation.

biophysics↗