Biology subjects

Pantoja-Uceda, D.

Publications and source records attributed to Pantoja-Uceda, D..

6 recordsLinked to original sources

bioRxiv2022

Insight into Polyproline II Helical Bundle Stability and Folding from NMR Spectroscopic Characterization of the Snow Flea Antifreeze Protein Denatured State

The use of PPII helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. To get atomic level information on the conformational ensemble and entropy of the reduced denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at three experimental conditions. No significant populations of preferred secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation of the two disulfide bonds during nascent folding. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured /{beta} proteins both on fast ps/ns as well as slower s/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

biophysics↗

bioRxiv2021

SARS-CoV-2 Nsp8 N-terminal domain dimerizes and harbors autonomously folded elements

The SARS-CoV-2 Nsp8 protein is a critical component of the RNA replicase, as its N-terminal domain (NTD) anchors Nsp12, the RNA, and Nsp13. Whereas its C-terminal domain (CTD) structure is well resolved, there is an open debate regarding the conformation adopted by the NTD as it is predicted as disordered but found in a variety of complex-dependent conformations or missing from many other structures. Using NMR spectroscopy, we show that the SARS CoV-2 Nsp8 NTD features both well folded secondary structure and disordered segments. Our results suggest that while part of this domain corresponding to two long -helices forms autonomously, the folding of other segments would require interaction with other replicase components. When isolated, the -helix population progressively declines towards the C-termini, and dynamics measurements indicate that the Nsp8 NTD behaves as a dimer under our conditions.

biophysics↗

bioRxiv2020

Glycine Rich Segments Adopt Polyproline II Helices Which May Contribute to Biomolecular Condensate Formation

Many intrinsically disordered proteins contain Gly-rich regions which are generally assumed to be disordered. Such regions often form biomolecular condensates which play essential roles in organizing cellular processes. However, the bases of their formation and stability are still not completely understood. Considering NMR studies of the Gly-rich H. harveyi "snow flea" antifreeze protein, we recently proposed that Gly-rich sequences, such as the third "RGG" region of Fused in Sarcoma (FUS) protein, may adopt polyproline II helices whose association might stabilize condensates. Here, this hypothesis is tested with a polypeptide corresponding to the third RGG region of FUS. NMR spectroscopy and molecular dynamics simulations suggest that significant populations of polyproline II helix are present. These findings are corroborated in a model peptide Ac-RGGYGGRGGWGGRGGY-NH2, where a peak characteristic of polyproline II helix is observed using CD spectroscopy. Its intensity suggests a polyproline II population of 40%. This result is supported by data from FTIR and NMR spectroscopies. In the latter, NOE correlations are observed between the Tyr and Arg, and Arg and Trp side chain hydrogens, confirming that side chains spaced three residues apart are close in space. Taken together, the data are consistent with a polyproline II helix, which is bent to optimize interactions between guanidinium and aromatic moieties, in equilibrium with a statistical coil ensemble. In cells, the polyproline II population of these peptides could be augmented by binding profilin protein or SH3, WW or OCRE domains, association with RNA or assembly into polyproline II helical bundles. These results lend credence to the hypothesis that Gly-rich segments of disordered proteins may form polyproline II helices which help stabilize biomolecular condensates.

biophysics↗

bioRxiv2020

Preferred Conformations in the Intrinsically Disordered Region of Human CPEB3 Explain its Role in Memory Consolidation

While implicated in neurodegenerative diseases, amyloids are also essential to some physiological processes, including memory consolidation by neuronal-specific isoforms of the Cytoplasmic Polyadenylation Element Binding (CPEB) protein family. CPEB mediates memory persistence by the formation of self-sustaining amyloid assemblies through its intrinsically disordered region (IDR). Here, we characterize the atomic level conformation and ps-ns dynamics of the 426-residue IDR of human CPEB3 (hCPEB3), which has been associated with episodic memory in humans, by NMR spectroscopy. We found that the first 29 residues: M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical+disordered motif. Residues 86-93: P83QQPPPP93, and 166-175: P166PPPAPAPQP175 form polyproline II (PPII) helices. While the (VG)5 repeat motif is completely disordered, residues 200-250 adopt three partially populated -helices. Residues 345-355, which comprise the nuclear localization signal (NLS), form a modestly populated -helix and border a phosphoTyr which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids. Besides revealing some aspects of the molecular basis of memory, these findings could aid the future development of treatments for post-traumatic stress disorder. Areas: Biophysics, Structural Biology, Biochemistry & Neurosciences.

biochemistry↗

bioRxiv2020

Phe-Gly Motifs Drive Fibrillization of TDP-43's Prion-Like Domain Droplets

TDP-43 assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g. muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct endpoint species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs, and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions, and illustrate structural features that distinguishes between aggregate forms.

biophysics↗

bioRxiv2019

Conformational Priming of RepA-WH1 for Functional Amyloid Conversion Detected by NMR Spectroscopy

How proteins with a stable globular fold acquire the amyloid state is still largely unknown. RepA is a versatile plasmidic DNA binding protein, functional either as a transcriptional repressor or as an initiator or inhibitor of DNA replication, the latter through the assembly of an amyloidogenic oligomer. Its N-terminal domain (WH1) is responsible for discrimination between these functional abilities by undergoing hitherto unknown structural changes. Furthermore, when expressed alone, RepA-WH1 behaves as a synthetic prion-like protein causing an amyloid proteinopathy in bacteria. RepA-WH1 is a stable dimer whose conformational dynamics had not been explored. Here we have studied it through NMR {1H}-15N relaxation and H/D exchange kinetics measurements. The N- and the C- terminal -helices, which lock the WH1 fold in each subunit of the dimer, as well as an internal amyloidogenic loop, show reduced stability and are partially unfolded in solution. S4-indigo, a small molecule ligand known to interfere with the amyloid assembly of RepA-WH1, binds to and tethers the N-terminal -helix and a {beta}-hairpin that is involved in dimerization, thus providing evidence for a priming role of fraying ends and dimerization switches in the amyloidogenesis of folded proteins.

biochemistry↗