Biology subjects

Parracino, A.

Publications and source records attributed to Parracino, A..

3 recordsLinked to original sources

bioRxiv2026

Hydration and H/D exchange-dependent infrared signatures of the GCN4 leucine zipper

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

biochemistry↗

bioRxiv2026

DNA supercoiling modulates bZIP transcription factor/DNA interaction

DNA topology is a key regulator of chromatin structure and transcription, yet its direct role in transcription factor recognition remains unclear. Here, we investigate how distinct DNA topological states modulate binding of the Saccharomyces cerevisiae bZIP transcription factor GCN4 using topologically defined plasmids. By combining, complementary biochemical approaches, including Bio-Layer Interferometry applied here for the first time to topology-dependent protein-DNA interactions, we show that DNA supercoiling directly reshapes GCN4-DNA recognition. Positively supercoiled DNA forms more stable and persistent complexes, whereas negatively supercoiled DNA retains greater conformational heterogeneity. To interpret these effects, we performed multiscale molecular simulations. Coarse-grained simulations of plasmids recapitulate the global topology-dependent trends observed experimentally, while matched minicircle models reproduce the same behaviour at the local scale. In strong agreement with experimental data, simulations reveal that DNA topology modulates the conformational ensemble of the GCN4 basic region. Overall, positively supercoiled DNA promotes a more ordered binding mode and localized protein distribution, whereas negatively supercoiled DNA supports increased structural plasticity. These findings identify DNA topology as an active determinant of transcription factor recognition and provide a multiscale framework linking global DNA mechanics to local protein-DNA interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/722604v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@18f8ba9org.highwire.dtl.DTLVardef@11a395dorg.highwire.dtl.DTLVardef@ac093borg.highwire.dtl.DTLVardef@923212_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Redefining the Limits of Functional Continuity in the Early Evolution of P-Loop NTPases

At the heart of many nucleoside triphosphatases is a conserved sequence motif that binds phosphate. A current model of early enzyme evolution proposes that this 6-8 residue motif could have sparked the emergence of the very first nucleoside triphosphatases - a striking example of evolutionary continuity from simple beginnings, if true. To test whether this provocative evolutionary model holds for the ancient and ubiquitous P-Loop NTPases, the properties of seven disembodied Walker A-derived peptides were extensively characterized by Hamiltonian replica exchange molecular dynamics simulations. Although dynamic flickers of nest-like conformations were observed, significant structural similarity between the situated peptide and its disembodied counterpart was not detected - even in the presence of orthophosphate or a nucleotide. Simulations suggest that phosphate binding is non-specific, with a slight preference for GTP over orthophosphate. Control peptides with the same amino acid composition but different sequences and situated conformations behaved similarly to the Walker A peptides with respect to conformational dynamics and phosphate binding, revealing no indication that the Walker A sequence is privileged as a disembodied peptide. We conclude that the evolutionary history of the P-Loop NTPase family is unlikely to have started with a disembodied Walker A peptide in an aqueous environment. The limits of evolutionary continuity for this protein family, and the environmental context within which it emerged, must be reconsidered. Finally, we argue that motifs such as the Walker A motif may represent incomplete or fragmentary molecular fossils - the true nature of which have been eroded by time. Significance StatementThe first proteins were undoubtedly small, but when did those seeds emerge, and what did they look like? It is widely believed that the Walker A P-loop motif is a seed for the emergence of phosphate binding proteins, snugly binding phosphate in a structurally conserved nest. We probe this hypothesis through detailed computational characterization of disembodied Walker A and control peptides, showing that any nest formation is transient, and phosphate binding is weak and non-specific. Thus, we do not find structural continuity represented in the conserved Walker A motif, and current models of early P-loop evolution require revision. Further, care is required when interpreting highly conserved sequence fragments more broadly, as these may merely represent eroded molecular fossils.

biophysics↗