Browsing by Author "Whitford, Paul C."

Now showing 1 - 7 of 7
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    Biomolecular dynamics: order–disorder transitions and energy landscapes
    (IOP Publishing, 2012) Whitford, Paul C.; Sanbonmatsu, Karissa Y.; Onuchic, José N.; Center for Theoretical Biological Physics
    While the energy landscape theory of protein folding is now a widely accepted view for understanding how relatively-weak molecular interactions lead to rapid and cooperative protein folding, such a framework must be extended to describe the large-scale functional motions observed in molecular machines. In this review, we discuss 1) the development of the energy landscape theory of biomolecular folding, 2) recent advances towards establishing a consistent understanding of folding and function, and 3) emerging themes in the functional motions of enzymes, biomolecular motors, and other biomolecular machines. Recent theoretical, computational, and experimental lines of investigation are providing a very dynamic picture of biomolecular motion. In contrast to earlier ideas, where molecular machines were thought to function similarly to macroscopic machines, with rigid components that move along a few degrees of freedom in a deterministic fashion, biomolecular complexes are only marginally stable. Since the stabilizing contribution of each atomic interaction is on the order of the thermal fluctuations in solution, the rigid body description of molecular function must be revisited. An emerging theme is that functional motions encompass order-disorder transitions and structural flexibility provide significant contributions to the free-energy. In this review, we describe the biological importance of order-disorder transitions and discuss the statistical-mechanical foundation of theoretical approaches that can characterize such transitions.
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    Design and proof of concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain
    (National Academy of Sciences, 2021) Staquicini, Daniela I.; Tang, Fenny H.F.; Markosian, Christopher; Yao, Virginia J.; Staquicini, Fernanda I.; Dodero-Rojas, Esteban; Contessoto, Vinícius G.; Davis, Deodate; O’Brien, Paul; Habib, Nazia; Smith, Tracey L.; Bruiners, Natalie; Sidman, Richard L.; Gennaro, Maria L.; Lattime, Edmund C.; Libutti, Steven K.; Whitford, Paul C.; Burley, Stephen K.; Onuchic, José Nelson; Arap, Wadih; Pasqualini, Renata; Center for Theoretical Biological Physics
    Development of effective vaccines against coronavirus disease 2019 (COVID-19) is a global imperative. Rapid immunization of the entire human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and different vaccine approaches are being pursued. Engineered filamentous bacteriophage (phage) particles 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 development and initial evaluation of two targeted phage-based vaccination approaches against SARS-CoV-2: dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. For peptide-targeted phage, we performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein. One of these epitopes displayed on the major capsid protein pVIII of phage induced a specific and sustained humoral response when injected in mice. These phage were further engineered to simultaneously display the peptide CAKSMGDIVC on the minor capsid protein pIII to enable their transport from the lung epithelium into the systemic circulation. Aerosolization of these “dual-display” phage into the lungs of mice generated a systemic and specific antibody response. In the second approach, targeted AAVP particles were engineered to deliver the entire S protein gene under the control of a constitutive CMV promoter. This induced tissue-specific transgene expression, stimulating a systemic S protein-specific antibody response in mice. With these proof-of-concept preclinical experiments, we show that both targeted phage- and AAVP-based particles serve as robust yet versatile platforms that can promptly yield COVID-19 vaccine prototypes for translational development.
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    Generalized Manning Condensation Model Captures the RNA Ion Atmosphere
    (American Physical Society, 2015) Hayes, Ryan L.; Noel, Jeffrey K.; Mandic, Ana; Whitford, Paul C.; Sanbonmatsu, Karissa Y.; Mohanty, Udayan; Onuchic, José N.; Center for Theoretical Biological Physics
    RNA is highly sensitive to the ionic environment and typically requires Mg2+ to form compact structures. There is a need for models capable of describing the ion atmosphere surrounding RNA with quantitative accuracy. We present a model of RNA electrostatics and apply it within coarse-grained molecular dynamics simulation. The model treats Mg2+ ions explicitly to account for ion-ion correlations neglected by mean-field theories. Since mean-field theories capture KCl well, it is treated implicitly by a generalized Manning counterion condensation model. The model extends Manning condensation to deal with arbitrary RNA conformations, nonlimiting KCl concentrations, and the ion inaccessible volume of RNA. The model is tested against experimental measurements of the excess Mg2+ associated with the RNA, Γ2+, because Γ2+ is directly related to the Mg2+-RNA interaction free energy. The excellent agreement with experiment demonstrates that the model captures the ionic dependence of the RNA free energy landscape.
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    Magnesium Fluctuations Modulate RNA Dynamics in the SAM-I Riboswitch
    (American Chemical Society, 2012) Hayes, Ryan L.; Noel, Jeffrey K.; Mohanty, Udayan; Whitford, Paul C.; Hennelly, Scott P.; Onuchic, José N.; Sanbonmatsu, Karissa Y.; Center for Theoretical Biological Physics
    Experiments demonstrate that Mg2+ is crucial for structure and function of RNA systems, yet the detailed molecular mechanism of Mg2+ action on RNA is not well understood. We investigate the interplay between RNA and Mg2+ at atomic resolution through ten 2-μs explicit solvent molecular dynamics simulations of the SAM-I riboswitch with varying ion concentrations. The structure, including three stemloops, is very stable on this time scale. Simulations reveal that outer-sphere coordinated Mg2+ ions fluctuate on the same time scale as the RNA, and that their dynamics couple. Locally, Mg2+ association affects RNA conformation through tertiary bridging interactions; globally, increasing Mg2+ concentration slows RNA fluctuations. Outer-sphere Mg2+ ions responsible for these effects account for 80% of Mg2+ in our simulations. These ions are transiently bound to the RNA, maintaining interactions, but shuttled from site to site. Outer-sphere Mg2+ are separated from the RNA by a single hydration shell, occupying a thin layer 3–5 Å from the RNA. Distribution functions reveal that outer-sphere Mg2+ are positioned by electronegative atoms, hydration layers, and a preference for the major groove. Diffusion analysis suggests transient outer-sphere Mg2+ dynamics are glassy. Since outer-sphere Mg2+ ions account for most of the Mg2+ in our simulations, these ions may change the paradigm of Mg2+–RNA interactions. Rather than a few inner-sphere ions anchoring the RNA structure surrounded by a continuum of diffuse ions, we observe a layer of outer-sphere coordinated Mg2+ that is transiently bound but strongly coupled to the RNA.
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    The Shadow Map: A General Contact Definition for Capturing the Dynamics of Biomolecular Folding and Function
    (American Chemical Society, 2012) Noel, Jeffrey K.; Whitford, Paul C.; Onuchic, José N.; Center for Theoretical Biological Physics
    Structure-based models (SBMs) are simplified models of the biomolecular dynamics that arise from funneled energy landscapes. We recently introduced an all-atom SBM that explicitly represents the atomic geometry of a biomolecule. While this initial study showed the robustness of the all-atom SBM Hamiltonian to changes in many of the energetic parameters, an important aspect, which has not been explored previously, is the definition of native interactions. In this study, we propose a general definition for generating atomically grained contact maps called “Shadow”. The Shadow algorithm initially considers all atoms within a cutoff distance and then, controlled by a screening parameter, discards the occluded contacts. We show that this choice of contact map is not only well behaved for protein folding, since it produces consistently cooperative folding behavior in SBMs but also desirable for exploring the dynamics of macromolecular assemblies since, it distributes energy similarly between RNAs and proteins despite their disparate internal packing. All-atom structure-based models employing Shadow contact maps provide a general framework for exploring the geometrical features of biomolecules, especially the connections between folding and function.
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    SMOG 2: A Versatile Software Package for Generating Structure-Based Models
    (Public Library of Science, 2016) Noel, Jeffrey K.; Levi, Mariana; Raghunathan, Mohit; Lammert, Heiko; Hayes, Ryan L.; Onuchic, José N.; Whitford, Paul C.; Center for Theoretical Biological Physics
    Molecular dynamics simulations with coarse-grained or simplified Hamiltonians have proven to be an effective means of capturing the functionally important long-time and large-length scale motions of proteins and RNAs. Originally developed in the context of protein folding, structure-based models (SBMs) have since been extended to probe a diverse range of biomolecular processes, spanning from protein and RNA folding to functional transitions in molecular machines. The hallmark feature of a structure-based model is that part, or all, of the potential energy function is defined by a known structure. Within this general class of models, there exist many possible variations in resolution and energetic composition. SMOG 2 is a downloadable software package that reads user-designated structural information and user-defined energy definitions, in order to produce the files necessary to use SBMs with high performance molecular dynamics packages: GROMACS and NAMD. SMOG 2 is bundled with XML-formatted template files that define commonly used SBMs, and it can process template files that are altered according to the needs of each user. This computational infrastructure also allows for experimental or bioinformatics-derived restraints or novel structural features to be included, e.g. novel ligands, prosthetic groups and post-translational/transcriptional modifications. The code and user guide can be downloaded at http://smog-server.org/smog2.
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    Substrate-Specific Reorganization of the Conformational Ensemble of CSK Implicates Novel Modes of Kinase Function
    (Public Library of Science, 2012) Jamros, Michael A.; Oliveira, Leandro C.; Whitford, Paul C.; Onuchic, José N.; Adams, Joseph A.; Jennings, Patricia A.; Center for Theoretical Biological Physics
    Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.