Biology subjects

Stan, G.

Publications and source records attributed to Stan, G..

8 recordsLinked to original sources

bioRxiv2026

Building from the ground up: augmentation strategies to improve protein language model performance on protein sets

An important underlying requirement for deep learning models is the availability of large datasets, however, in sciences such as chemistry and biology, generating experimental data of sufficient size represents a significant bottleneck. In this work, we probed the extent and nature of limited input data, characterize how several protein language models (pLMs) learned on suboptimal datasets, and investigated augmentation approaches to improve predictive model performance. We found that narrowing the distribution of protein-protein complexes in a limited dataset permits an order-of-magnitude decrease in training dataset size, if a model is only expected to generalize to in-distribution data. Similar results were found in active learning (AL), where both in- and out-of-distribution Matthews Correlation Coefficient (MCC) showed a small but consistent increase upon augmentation of the training set with complexes produced by ESM-3, a generative pLM.

biophysics↗

bioRxiv2026

Probing Mechanisms of Allosteric Regulation in AAA+ ATPases for Microtubule Severing and Protein Disaggregation

Ring-like AAA+ (ATPases Associated with diverse cellular Activities) biological machines mediate protein remodeling to assist a broad range of essential cellular functions. The nucleotide-dependent remodeling action involves intra- and inter-ring allosteric communication to generate mechanical force applied onto the substrate by a set of loops that protrude into the central channel. In this study, we probe these allosteric mechanisms through a comparative study of the katanin, a microtubule severing protein including a clade 3 AAA domain, and the double-ring ClpB, a protein disaggregase including both a clade 3 and a clade 5 AAA domain. Our molecular dynamics simulations, combined with machine learning and bioinformatic analysis, reveal both similar mechanisms involving the clade 3 domain and ClpB-specific ones involving communication with the clade 5 domain. We find that both nucleotide and substrate polypeptide binding restrict the conformational landscape sampled by katanin and ClpB, with ligand-specific conformations observed in the latter case. Allosteric contributions of secondary structure elements, ranked by using SHapley Additive exPlanations analysis in machine learning approaches and binary classification of features in ligand states, highlight the important role of regions adjacent to the nucleotide-binding site and the pore loops. Amino acid-level analysis of the allosteric paths reveals that intra-ring cooperativity modulates long-distance communication within the AAA+ protomers.

biophysics↗

bioRxiv2026

Vessel Organoids Reveal FOXF1 Variant-Specific Regulation of Mesoderm and Capillary Development

How allelic variants in lineage-regulating transcription factors drive diverging human developmental outcomes remains poorly understood. This is partly due to the lack of human model systems. Here, we used vessel organoids from human induced pluripotent stem cells (hiPSCs) to resolve variant-specific functions of Forkhead Box F1 (FOXF1), a critical regulator of mesoderm and vascular development. Using three patient-derived hiPSC lines harboring unique FOXF1 variants, we show that heterozygous variants cause capillary maldevelopment of varying severity. Single-nucleus multiomic analysis revealed variant-specific mechanisms - a severe variant impairs differentiation of nascent mesoderm to lateral plate mesoderm and disrupts vascular progenitor specification, while moderate variants permit mesoderm differentiation but rewire vascular progenitor states and function. Restoration of wild-type FOXF1 via lipid nanoparticle-mediated mRNA delivery rescued capillary formation in a variant- and developmental-stage-dependent manner. Together, these findings demonstrate that different variants disrupt stage-specific FOXF1 functions in human mesoderm-to-vascular development, underscoring the importance of variant-specific therapeutic strategies. HIGHLIGHTS O_LIHuman vessel organoids reveal variant-specific roles of FOXF1 in mesoderm patterning and capillary development. C_LIO_LISevere FOXF1 variant c.253T>A (p.F85I) impairs nascent mesoderm-to-lateral plate mesoderm differentiation and disrupts vascular progenitor specification. C_LIO_LI Moderate FOXF1 variants differentially rewire endothelial and mural progenitor cell states and function. C_LIO_LILipid nanoparticle-mediated FOXF1 mRNA delivery rescues capillary formation in a variant- and developmental-stage-dependent manner. C_LI

developmental biology↗

bioRxiv2023

Allosteric Communication in the Gating Mechanism for Controlled Protein Degradation by the Bacterial ClpP Peptidase

Proteolysis is essential for the control of metabolic pathways and cell cycle. Bacterial caseinolytic proteases (Clp) use peptidase components, such as ClpP, to degrade defective substrate proteins and to regulate cellular levels of stress-response proteins. To ensure selective degradation, access to the proteolytic chamber of the double- ring ClpP tetradecamer is controlled by a critical gating mechanism of the two axial pores. Binding of conserved loops of the Clp ATPase component of the protease or small molecules, such as acyldepsipeptide (ADEP), at peripheral ClpP ring sites triggers axial pore opening through dramatic conformational transitions of flexible N-terminal loops between disordered conformations in the "closed" pore state and ordered hairpins in the "open" pore state. In this study, we probe the allosteric communication underlying these conformational changes by comparing residue-residue couplings in molecular dynamics simulations of each configuration. Both principal component and normal mode analyses highlight large-scale conformational changes in the N-terminal loop regions and smaller amplitude motions of the peptidase core. Community network analysis reveals a switch between intraand inter-protomer coupling in the open - close pore transition. Allosteric pathways that connect the ADEP binding sites to N-terminal loops are rewired in this transition, with shorter network paths in the open pore configuration supporting stronger intra- and inter-ring coupling. Structural perturbations, either through removal of ADEP molecules or point mutations, alter the allosteric network to weaken the coupling.

biophysics↗

bioRxiv2023

Single-molecule FRET probes the allosteric effect of ATP on the protein-translocating pore loops of a AAA+ machine

AAA+ proteins (ATPases associated with various cellular activities) comprise a family of powerful ring-shaped ATP-dependent translocases that carry out numerous vital substrate-remodeling functions. ClpB is a AAA+ protein disaggregation machine that forms a two-tiered hexameric ring, with flexible pore loops protruding into its center and binding to substrate-proteins. It remains unknown whether these pore loops contribute only passively to substrate-protein threading or have a more active role. Recently, we have applied single-molecule FRET (smFRET) spectroscopy to directly measure the dynamics of substrate-binding pore loops in ClpB. We have reported that the three pore loops of ClpB (PL1-3) undergo large-scale fluctuations on the microsecond timescale that are likely to be mechanistically important for disaggregation. Here, using smFRET, we study the allosteric coupling between the pore loops and the two nucleotide binding domains of ClpB (NBD1-2). By mutating the conserved Walker B motifs within the NBDs to abolish ATP hydrolysis, we demonstrate how the nucleotide state of each NBD tunes pore loop dynamics. This effect is surprisingly long-ranged; in particular, PL2 and PL3 respond differentially to a Walker B mutation in either NBD1 or NBD2, as well as to mutations in both. We characterize the conformational dynamics of pore loops and the allosteric paths connecting NBDs to pore loops by molecular dynamics simulations and find that both principal motions and allosteric paths can be altered by changing the ATPase state of ClpB. Remarkably, PL3, which is highly conserved in AAA+ machines, is found to favor an upward conformation when only NBD1 undergoes ATP hydrolysis, but a downward conformation when NBD2 is active. These results explicitly demonstrate a significant long-range allosteric effect of ATP hydrolysis sites on pore-loop dynamics. Pore loops are therefore established as active participants that undergo ATP-dependent conformational changes to translocate substrate proteins through the central pores of AAA+ machines. Statement of SignificanceMolecular machines function by coupling the energy of ATP hydrolysis to mechanical motion. How this coupling occurs and what timescales are involved remains an open question. In this study, we use a powerful single-molecule FRET technique to measure the real-time dynamics of pore loops, which are essential protein-translocating elements of the ATP-dependent disaggregation machine ClpB. Using a series of mutations of the ATP-hydrolysis motifs of ClpB, we find that, although the motions of these pore loops take place on the microsecond time scale, they are markedly affected by the much slower changes in the nucleotide state of the machine. Generally, this study shows that protein machines, such as ClpB, are wired to harness ATP binding and hydrolysis to allosterically affect distal events, such as the function-related mechanics of pore-loops.

biophysics↗

bioRxiv2022

Exploring the effect of mechanical anisotropy of protein structures in the unfoldase mechanism of AAA+ molecular machines

Essential cellular processes of microtubule disassembly and protein degradation, which span lengths from tens of m to nm, are mediated by specialized molecular machines with similar hexameric structure and function. Our molecular simulations at atomistic and coarse-grained scales show that both the microtubule severing protein spastin and the caseinolytic protease ClpY, accomplish spectacular unfolding of their diverse substrates, a microtubule lattice and dihydrofolate reductase (DHFR), by taking advantage of mechanical anisotropy in these proteins. By considering wild-type and variants of DHFR, we found that optimal ClpY-mediated action probes favorable orientations of the substrate relative to the machine. Unfolding of wild-type DHFR involves strong mechanical interfaces near each terminal and occurs along branched pathways, whereas unfolding of DHFR variants involves softer mechanical interfaces and occurs through single pathways, but translocation hindrance can arise from internal mechanical resistance. For spastin, optimum severing action initiated by pulling on a tubulin subunit is achieved through the orientation of the machine versus the substrate (microtubule lattice). Moreover, changes in the strength of the interactions between spastin and a microtubule filament, which can be driven by the tubulin code, lead to drastically different outcomes for the integrity of the hexameric structure of the machine.

biophysics↗

bioRxiv2021

Unfolding and Translocation of Knotted Proteins by Clp Biological Nanomachines: Synergistic Contribution of Primary Sequence and Topology Revealed by Molecular Dynamics Simulations

We use Langevin dynamics simulations to model, at atomistic resolution, how various natively-knotted proteins are unfolded in repeated allosteric translocating cycles of the ClpY ATPase. We consider proteins representative of different topologies, from the simplest knot (trefoil 31), to the three-twist 52 knot, to the most complex stevedore, 61, knot. We harness the atomistic detail of the simulations to address aspects that have so far remained largely unexplored, such as sequence-dependent effects on the ruggedness of the landscape traversed during knot sliding. Our simulations reveal the combined effect on translocation of the knotted protein structure, i.e. backbone topology and geometry, and primary sequence, i.e. side chain size and interactions, and show that the latter can even dominate translocation hindrance. In addition, we observe that, due to the interplay between the knotted topology and intramolecular contacts, the transmission of tension along the peptide chain occurs very differently from homopolymers. Finally, by considering native and non-native interactions, we examine how the disruption or formation of such contacts can affect the translocation processivity and concomitantly create multiple unfolding pathways with very different activation barriers.

biophysics↗

bioRxiv2020

Factors Underlying Asymmetric Dynamics of Disaggregase and Microtubule Severing AAA+ Machines

Disaggregation and microtubule-severing nanomachines from the AAA+ (ATPases associated with various cellular activities) superfamily assemble into ring-shaped hexamers that enable protein remodeling by coupling large-scale conformational changes with application of mechanical forces within a central pore by loops protruding within the pore. We probed these motions and intra-ring interactions that support them by performing extensive explicit solvent molecular dynamics simulations of single-ring severing proteins and the double-ring disaggregase ClpB. Simulations reveal that dynamic stability of hexamers of severing proteins and of the nucleotide binding domain 1 (NBD1) ring of ClpB, which belong to the same clade, involves a network of salt bridges that connect conserved motifs of central PL1 loops of the hexamer. Clustering analysis of ClpB highlights correlated motions of domains of neighboring protomers supporting strong inter-protomer collaboration. Severing proteins have weaker inter-protomer coupling and stronger intra-protomer stabilization through salt bridges formed between PL2 and PL3 loops. Distinct mechanisms are identified in the NBD2 ring of ClpB involving weaker inter-protomer coupling through salt bridges formed by non-canonical loops and stronger intra-protomer coupling. Pore width fluctuations associated with the PL1 constriction in the spiral states, in the presence of a substrate peptide, highlight stark differences between narrowing of channels of severing proteins and widening of the NBD1 ring of ClpB. This indicates divergent substrate processing mechanisms of remodeling and translocation by ClpB and substrate tail-end gripping and possible wedging on microtubule lattice by severing enzymes. Relaxation dynamics of the distance between the PL1 loops and the centers of mass of protomers reveals observation-time-dependent dynamics, leading to predicted relaxation times of tens of microseconds on millisecond experimental timescales. For ClpB the predicted relaxation time is in excellent agreement with the extracted time from smFRET experiments.

biophysics↗