Biology subjects

Hilser, V. J.

Publications and source records attributed to Hilser, V. J..

6 recordsLinked to original sources

bioRxiv2026

Multivalent weak contacts shape chaperone-nascent protein interactions

Molecular chaperones interact with non-native proteins, playing crucial roles in preventing misfolding and enable efficient folding in the cellular environment. Trigger factor is a bacterial chaperone that binds to ribosomes, interacting with nascent polypeptides emerging from the ribosome and guiding their early folding steps. In contrast to the central role of the chaperone in promoting folding of newly synthesized proteins, its dynamic interactions with nascent chains emerging from the ribosome remain poorly understood. Here, we use single-molecule fluorescence and optical tweezers approaches to directly observe and characterize trigger factor interactions with a ribosome-bound client protein at increasing chain lengths. We find that trigger factor binding to nascent proteins is best described by a combination of multiple weak, dynamic interactions that are established after the chaperone docks onto the ribosome and evolve during polypeptide elongation. Application of mechanical force perturbs trigger factor binding, supporting a multivalent interaction model. This binding mode may help to stabilize nascent proteins against misfolding while allowing them to dynamically sample conformational space in search of their native structures.

biophysics↗

bioRxiv2025

Chaperone saturation mediates translation and protein folding efficiency

Whether the emergence of a nascent protein from the ribosome and the formation of structural elements are synchronized has been a longstanding question (Chaney and Clark, 2015; Deane and Saunders, 2011; Levinthal, 1968; Marin, 2008; Sauna and Kimchi-Sarfaty, 2011; Spencer and Barral, 2012; Tsai et al., 2008; Zhang and Ignatova, 2011). Paradoxically, kinetically efficient translation can induce mis-folding and aggregation despite the presence of molecular chaperones (Siller et al., 2010; Spencer et al., 2012), which in Escherichia coli are induced by unfolded protein (Parsell and Sauer, 1989) via {sigma}32 (Craig and Gross, 1991). The molecular mechanisms mediating translation efficiency and protein folding efficiency remain poorly understood. Using ribosome profiling (Ingolia et al., 2009) and protein quantitation, we show that synonymous changes to Firefly Luciferase (Luc) mRNA have a direct effect on its translation efficiency. These changes alone cause up to a 70-fold difference in Luc protein levels. However, increased Luc protein is met with at most a [~]2-fold increase in chaperone levels, revealing that the {sigma}32 transcriptional response has saturable properties. This response is found to be poised near its midpoint (where it is most sensitive to perturbation) when Luc mRNA has an intermediate translation efficiency. These results suggest not only that chaperone saturation limits the ability of cells to maintain protein folding homeostasis when challenged with highly efficient translation, but that translation efficiency and protein folding efficiency evolved for mutual sensitivity.

systems biology↗

bioRxiv2023

The variable domain from the mitochondrial fission mechanoenzyme Drp1 promotes liquid-liquid phase separation

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" (VD) important for regulation. For the mitochondrial fission dynamin, Drp1, a regulatory role for the VD is demonstrated by mutations that can elongate, or fragment, mitochondria. How the VD encodes inhibitory and stimulatory activity is unclear. Here, isolated VD is shown to be intrinsically disordered (ID) yet undergoes a cooperative transition in the stabilizing osmolyte TMAO. However, the TMAO stabilized state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, raising the possibility that phase separation may enable rapid tuning of Drp1 assembly necessary for fission.

biophysics↗

bioRxiv2021

A Predictive Energy Landscape Model of MetamorphicProtein Conformational Specificity

"Metamorphic" proteins challenge state-of-the-art structure prediction methods reliant on amino acid similarity. Unfortunately, this obviates a more effective thermodynamic approach necessary to properly evaluate the impact of amino acid changes on the stability of two different folds. A vital capability of such a thermodynamic approach would be the quantification of the free energy differences between 1) the energy landscape minima of each native fold, and 2) each fold and the denatured state. Here we develop an energetic framework for conformational specificity, based on an ensemble description of protein thermodynamics. This energetic framework was able to successfully recapitulate the structures of high-identity enginerered sequences experimentally shown to adopt either Streptococcus protein GA or GB folds, demonstrating that this approach indeed reflected the energetic determinants of fold. Residue-level decomposition of the conformational specificity suggested several testable hypotheses, notably among them that fold-switching could be affected by local de-stabilization of the populated fold at positions sensitive to equilibrium perturbation. Since this ensemble-based compatibility framework is applicable to any structure and any sequence, it may be practically useful for the future targeted design, or large-scale proteomic detection, of novel metamorphic proteins. Impact StatementMetamorphic proteins are single amino acid sequences capable of adopting more than one structure at equilibrium. Detection and design of these molecules hold great promise for biological understanding and materials engineering, but to do so requires a thermodynamic framework capable of estimating the free energy differences between the two structures and the denatured state. We present such a framework, show it to be effective for the well-studied metamorphic protein GA/GB system, and suggest testable hypotheses for engineering novel fold-switch proteins.

biochemistry↗

bioRxiv2020

Tumor susceptibility gene-101 regulates glucocorticoid receptor through disorder-mediated allostery

Tumor Susceptibility Gene-101 (TSG101) is involved in endosomal maturation and has been implicated in the transcriptional regulation of several steroid hormone receptors (SHRs), although a detailed characterization of such regulation has yet to be conducted. Here we directly measure binding of TSG101 to one SHR, glucocorticoid receptor (GR). Using biophysical and cellular assays, we show that the coiled-coil domain of TSG101; 1) binds and folds the disordered N-terminal domain (NTD) of GR, 2) upon binding, improves DNA-binding of GR in vitro, and 3) enhances the transcriptional activity of GR in vivo. Our findings suggest that TSG101 is a bona fide transcriptional co-regulator of GR.

biophysics↗

bioRxiv2019

Hidden dynamic signatures drive substrate selectivity in the disordered phosphoproteome

Phosphorylation sites are hyper-abundant in the disordered proteins of eukaryotes, suggesting that conformational dynamics (or heterogeneity) may play a major role in determining to what extent a kinase interacts with a particular substrate. In biophysical terms, substrate selectivity may be determined not just by the structural and chemical complementarity between the kinase and its protein substrates, but also by the free energy difference between the conformational ensembles that are recognized by the kinase and those that are not. To test this hypothesis, we developed an informatics framework based on statistical thermodynamics, which allows us to probe for dynamic contributions to phosphorylation, as evaluated by the ability to predict Ser/Thr/ Tyr phosphorylation sites in the disordered proteome. Essential to this framework is a decomposition of substrate sequence information into two types: vertical information encoding conserved kinase specificity motifs and horizontal (distributed) information encoding substrate conformational dynamics that are embedded, but often not apparent, within position specific conservation patterns. We find not only that conformational dynamics play a major role, but that they are the dominant contribution to substrate selectivity. In fact, the main substrate classifier distinguishing selectivity is the magnitude of change in compaction of the disordered chain upon phosphorylation. Thus, in addition to providing fundamental insights into the underlying mechanistic consequences of phosphorylation across the entire proteome, our approach provides a novel statistical thermodynamic strategy for partitioning any sequence-based search into contributions from direct chemical and structural complementarity and those from changes in conformational dynamics. Using this framework, we developed a high-performance open-source phosphorylation site predictor, PHOSforUS, which is freely available at https://github.com/bxlab/PHOSforUS.

biophysics↗