Biology subjects

Whitford, P. C.

Publications and source records attributed to Whitford, P. C..

14 recordsLinked to original sources

bioRxiv2026

Molecular sterics guide tmRNA translocation on the ribosome

Translation of defective mRNA transcripts can lead to the formation of stalled ``non-stop'' ribosome complexes. To rescue these ribosomes, bacteria have evolved a mechanism called trans-translation that is mediated by transfer-messenger RNA (tmRNA) and small protein B (SmpB). During the translocation step of trans-translation, the stalled mRNA is ejected and the reading frame found in the mRNA-like domain (MLD) of tmRNA is loaded into the mRNA channel. The ribosome then translates the new template, thereby tagging the nascent protein for degradation. While structures of tmRNA-SmpB on the ribosome have been available for years, little is known about the molecular factors that govern the dynamics of tmRNA translocation. Here, we use an energetically smooth model (i.e. SMOG) to simulate translocation of tmRNA-SmpB, which reveals that the structure of the ribosome gives rise to a preferred sequence of conformational substeps. Specifically, the tRNA-like domain (TLD) translocates, then the MLD loads, and finally the C-terminal tail of SmpB rearranges. These steps are interdependent and associated with a range of sterically-induced barriers that regulate the kinetics. This provides a structural and energetic framework for understanding, and ultimately controlling, the dynamics of trans-translation.

biophysics↗

bioRxiv2026

Asynchronous structural rearrangements govern constriction of the human nuclear pore complex

The nuclear pore complex (NPC) is responsible for transport between the cytoplasm and nucleus. CryoET experiments have resolved multiple conformations of the pore that are dilated or constricted. Since the dilation state will impact transport properties, there is much interest in understanding the energetic factors that regulate dilation/constriction. To this end, we applied a structure-based (G[o]-like) model to study the mechanistic properties of large-scale constriction ( [~] 100[A]) in the human NPC. Even though the structures are symmetric, the simulations implicate asynchronous rear-rangements during constriction. By considering variants of the model, we further characterize the relative contributions of individual proteins to this collective process. Together, this provides an energetic/structural foundation that can guide development of precise physical approaches for studying this elusive motion.

biophysics↗

bioRxiv2025

Multi-Scale Kinetics Modeling and Advanced Assay for mRNA-Lipid Nanoparticle Potency Assessment

mRNA lipid nanoparticle (mRNA-LNP) technology has attracted global attention, especially in vaccine development, due to its superior delivery efficiency, molecular stability, and safety profile. However, evaluating mRNA-LNP potency--defined as the quantifiable biological response elicited by the product--remains costly and time-consuming when relying solely on in vitro experiments. Rapid and reliable potency assessment is hindered by limited mechanistic understanding of delivery processes and sparse experimental data. To address these challenges, we present a mechanism-informed, multi-scale kinetic modeling framework that quantitatively captures the coupled dynamics across particle-level, cellular, and macroscopic scales. This model incorporates variability in LNP-cell interactions and accounts for critical factors such as dosage, LNP and cell size distributions, and cell proliferation dynamics--all of which influence delivery efficiency and response variability. By integrating advanced multi-omics assays--such as single-molecule fluorescent in situ hybridization (smFISH), which enables single-cell resolution of mRNA and protein expression--our framework leverages heterogeneous, multi-scale data to support mechanistically grounded modeling of mRNA delivery and enable robust predictions of therapeutic potency. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/679406v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1f3ec2borg.highwire.dtl.DTLVardef@1163893org.highwire.dtl.DTLVardef@1dc530borg.highwire.dtl.DTLVardef@1d0264a_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2025

Transient ion-mediated interactions regulate subunit rotation in a eukaryotic ribosome

While it is known that ions are required for folding of RNA, little is known about how transient/probabilistic ionic interactions facilitate biologically-relevant conformational rearrangements. To address this, we developed a theoretical model that employs all-atom resolution, with a simplified representation of biomolecular energetics, explicit electrostatics and ions (K+, Cl-, Mg2+). For well-studied RNA systems (58-mer and Ade riboswitch), the model accurately describes the concentration-dependent ionic environment, including (bidentate) chelated and hydrated (diffuse/outer-shell) ions. With this foundation, we applied the model to simulate the yeast ribosome and quantified the ion-dependent energy landscape of intersubunit rotation. These calculations show how the energetics of rotation responds to millimolar changes in [MgCl2], which shift the distribution between rotation states and alter the kinetics by more than an order of magnitude. We find that this response to the ionic concentration correlates with formation and breakage of ion-mediated interactions (inner-shell and outer-shell) between the ribosomal subunits. This analysis provides a physical basis for understanding how transient ion-mediated interactions can regulate a large-scale biological process.

biophysics↗

bioRxiv2025

Characterization of a potential antibiotic target site on the ribosome

The ribosome is a known antibiotic target, where various classes of small molecules impact different stages of translation. There are multiple modes of function, such as interfering with the decoding process, impeding movement of the nascent protein chain and occluding the catalytic center. In the present study, we used a range of computational methods to demonstrate a new mechanism by which small molecules may impede translation in the bacterial ribosome. Specifically, we use a computational screen to identify small molecules that can bind the ribosomal protein L33 in a region that is generally conserved in bacterial species. In addition, the binding position allows it to introduce a steric obstacle that impedes the P/E hybrid-state formation steps. Using molecular dynamics simulations, we show how binding to L33 can slow down the kinetics of tRNA molecules on the ribosome. Since L33 is not present in the cytosolic human ribosome and has a distinct sequence in the human mitochondrial ribosome, this binding site may serve as a novel target for future antibiotic and antimicrobial design efforts. This analysis provides insights into how to optimize the inhibitory effects of L33-targeting molecules, in order to develop a new class of broad-spectrum antibiotics/antimicrobials.

biophysics↗

bioRxiv2025

How subunit rotation controls tRNA dynamics in the ribosome

In order to translate messenger RNA into proteins, the ribosome must coordinate a wide range of conformational rearrangements. Some steps involve individual molecules, whereas others require synchronization of multiple collective motions. For example, the ribosomal "small" subunit ([~]1 MDa) is known to undergo rotational motion ([~] 10{whitebullet}) that is correlated with large-scale displacements of tRNA molecules ([~]50[A]). While decades of biochemical, single-molecule and structural analysis have provided many insights into the timing of these motions, little is known about how these dynamical processes influence each other. To address this, we use molecular simulations to isolate specific interactions that allow tRNA kinetics to be controlled by subunit rotation. Specifically, we applied an all-atom structure-based model to perform molecular simulations of P/E hybrid formation, a process by which the tRNA molecules are displaced between ribosomal binding sites. By varying the extent of subunit rotation in each simulation, these calculations reveal a pronounced non-monotonic dependence of tRNA kinetics on subunit rotation. That is, tRNA kinetics increase and then decrease as the subunit is rotated. We further show that this behavior is due to the steric contributions of a single ribosomal protein. Together, these calculations provide insights into the physicochemical properties of the ribosome, while establishing a strategy for isolating causal relationships in biomolecular assemblies.

biophysics↗

bioRxiv2022

Ratchet, swivel, tilt and roll: A complete description of subunit rotation in the ribosome

Protein synthesis by the ribosome involves large-scale rearrangements of the "small" subunit (SSU; [~]1 MDa), which include inter- and intra-subunit rotational motions. With more than 1000 structures of ribosomes and ribosomal subunits now publicly available, it is becoming increasingly difficult to design precise experiments that are based on a comprehensive analysis of all known rotation states. To overcome this limitation, we present the Ribosome Angle Decomposition (RAD) method, where the orientation of each small subunit head and body is described in terms of three angular coordinates (rotation, tilt and tilt direction) and a single translation. To demonstrate the utility of the accompanying software (RADtool) we applied it to all published ribosome and mitoribosome structures. This identified and analyzed 1077 fully-assembled ribosome complexes, as well as 280 isolated small subunits from 48 organisms. The RAD approach quantitatively distinguishes between previously described qualitative rotational features, determines when rotation-only descriptions are insufficient, and shows that tilt-like rearrangements of the SSU head and body are pervasive in both prokaryotic and eukaryotic ribosomes. Together, the presented database and technique provide a robust platform for systematically analyzing, visualizing, and comparing subunit orientations of ribosomes from all kingdoms of life. Accordingly, the RAD resource establishes a common foundation with which structural, simulation, single-molecule and biochemical efforts can precisely interrogate the dynamics of this prototypical molecular machine.

molecular biology↗

bioRxiv2021

Apropos of Universal Epitope Discovery for COVID-19 Vaccines: A Framework for Targeted Phage Display-Based Delivery and Integration of New Evaluation Tools

Targeted bacteriophage (phage) particles are potentially attractive yet inexpensive platforms for immunization. Herein, we describe targeted phage capsid display of an immunogenically relevant epitope of the SARS-CoV-2 Spike protein that is empirically conserved, likely due to the high mutational cost among all variants identified to date. This observation may herald an approach to developing vaccine candidates for broad-spectrum, towards universal, protection against multiple emergent variants of coronavirus that cause COVID-19.

microbiology↗

bioRxiv2021

SMOG 2 and OpenSMOG: Extending the limits of structure-based models

Applying simulations with structure-based (G[o]-like) models has proven to be an effective strategy for investigating the factors that control biomolecular dynamics. The common element of these models is that some (or all) of the intra/inter-molecular inter-actions are explicitly defined to stabilize an experimentally-determined structure. To facilitate the development and application of this broad class of models, we previously released the SMOG 2 software package. This suite allows one to easily customize and distribute structure-based (i.e. SMOG) models for any type of polymer-ligand system. Since its original release, user feedback has driven the implementation of numerous enhancements. Here, we describe recent extensions to the software and demonstrate the capabilities of the most recent version, SMOG v2.4. Changes include new tools that aid user-defined customization of force fields, as well as an interface with the OpenMM simulation libraries (OpenSMOG v1.0). To illustrate the utility of these advances, we present several applications of SMOG2 and OpenSMOG, which include systems with millions of atoms, long polymers and explicit ions. We also highlight how one can incorporate non-structure-based (e.g. AMBER-based) energetics to define a hybrid class of models. The representative applications include large-scale rearrangements of the SARS-CoV-2 Spike protein, the HIV-1 capsid in the presence of explicit ions, and crystallographic lattices of ribosomes and proteins. In summary, SMOG 2 and OpenSMOG provide robust support for researchers who seek to apply structure-based models to large and/or intricate biomolecular systems.

biophysics↗

bioRxiv2021

Diffuse ions coordinate dynamics in a ribonucleoprotein assembly

Proper ionic concentrations are required for the functional dynamics of RNA and ribonucleoprotein (RNP) assemblies. While experimental and computational techniques have provided many insights into the properties of chelated ions, less is known about the energetic contributions of diffuse ions to large-scale conformational rearrangements. To address this, we present a model that is designed to quantify the influence of diffuse monovalent and divalent ions on the dynamics of biomolecular assemblies. This model employs all-atom (non-H) resolution and explicit ions, where effective potentials account for hydration effects. We first show that the model accurately predicts the number of excess Mg2+ ions for prototypical RNA systems, at a level comparable to modern coarse-grained models. We then apply the model to a complete ribosome and show how the balance between diffuse Mg2+ and K+ ions can control the dynamics of tRNA molecules during translation. The model predicts differential effects of diffuse ions on the free-energy barrier associated with tRNA entry and the energy of tRNA binding to the ribosome. Together, this analysis reveals the direct impact of diffuse ions on the dynamics of an RNP assembly. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/448160v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@14f36caorg.highwire.dtl.DTLVardef@1963073org.highwire.dtl.DTLVardef@146fbf8org.highwire.dtl.DTLVardef@14860ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

The dynamics of subunit rotation in a eukaryotic ribosome

Protein synthesis by the ribosome is coordinated by an intricate series of large-scale conformational rearrangements. Structural studies can provide information about long-lived states, however biological kinetics are controlled by the intervening free-energy barriers. While there has been progress describing the energy landscapes of bacterial ribosomes, very little is known about the energetics of large-scale rearrangements in eukaryotic systems. To address this topic, we constructed an all-atom model with simplified energetics and performed simulations of subunit rotation in the yeast ribosome. In these simulations, the small subunit (SSU; ~1MDa) undergoes spontaneous and reversible rotations (~ 8{degrees}). By enabling the simulation of this rearrangement under equilibrium conditions, these calculations provide initial insights into the molecular factors that control dynamics in eukaryotic ribosomes. Through this, we are able to identify specific inter-subunit interactions that have a pronounced influence on the rate-limiting free-energy barrier. We also show that, as a result of changes in molecular flexibility, the thermodynamic balance between the rotated and unrotated states is temperature-dependent. This effect may be interpreted in terms of differential molecular flexibility within the rotated and unrotated states. Together, these calculations provide a foundation, upon which the field may begin to dissect the energetics of these complex molecular machines.

biophysics↗

bioRxiv2021

Design and proof-of-concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain

Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed "dual-display"), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development. SignificanceThe ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.

microbiology↗

bioRxiv2019

Dissecting the energetics of subunit rotation in the ribosome

The accurate expression of proteins requires the ribosome to efficiently undergo elaborate conformational rearrangements. The most dramatic of these motions is subunit rotation, which is necessary for tRNA molecules to transition between ribosomal binding sites. While rigid-body descriptions provide a qualitative picture of the process, obtaining quantitative mechanistic insights requires one to account for the relationship between molecular flexibility and collective dynamics. Using simulated rotation events, we assess the quality of experimentally-accessible measures for describing the collective displacement of the ~ 4000-residue small subunit. For this, we ask whether each coordinate is able to identify the underlying free-energy barrier and transition state ensemble (TSE). We find that intuitive structurally-motivated coordinates (e.g. rotation angle, inter-protein distances) can distinguish between the endpoints, though they are poor indicators of barrier-crossing events, and they underestimate the free-energy barrier. In contrast, coordinates based on inter-subunit bridges can identify the TSE. We additionally verify that the committor probability for the putative TSE configurations is 0.5, a hallmark feature of any transition state. In terms of structural properties, these calculations implicate a transition state in which flexibility allows for asynchronous rearrangements of the bridges as the ribosome adopts a partially-rotated orientation. These calculations provide a theoretical foundation, upon which experimental techniques may precisely quantify the energy landscape of the ribosome.

biophysics↗

bioRxiv2018

Using SMOG 2 to simulate complex biomolecular assemblies

Over the last 20 years, the application of structure-based (G[o]-like) models has ranged from protein folding with coarse-grained models to all-atom representations of large-scale molecular assemblies. While there are many variants that may be employed, the common feature of these models is that some (or all) of the stabilizing energetic interactions are defined based on knowledge of a particular experimentally-obtained conformation. With the generality of this approach, there was a need for a versatile computational platform for designing and implementing this class of models. To this end, the SMOG 2 software package provides an easy-to-use interface, where the user has full control of the model parameters. This software allows the user to edit XML-formatted files in order to provide definitions of new structure-based models. SMOG 2 reads these \"template\" files and maps the interactions onto specific structures, which are provided in PDB format. The force field files produced by SMOG 2 may then be used to perform simulations with a variety of popular molecular dynamics suites. In this chapter, we describe some of the key features of the SMOG 2 package, while providing examples and strategies for applying these techniques to complex (often large-scale) molecular assemblies, such as the ribosome.

biophysics↗