Browsing by Subject "Allostery"

Now showing 1 - 5 of 5
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    Data for Unbiased Clustering of Residues Undergoing Synchronous Motions in Proteins using NMR Spin Relaxation Data
    (2025-02-13) Veliparambil Subrahmanian, Manu; Veglia, Gianluigi; Melacini, Giuseppe; L Kovrigin, Evgenii; Loria, J Patrick; mvelipar@umn.edu; Veliparambil Subrahmanian, Manu; Veglia Lab
    Carr-Purcell-Meiboom-Gill Relaxation Dispersion (CPMG-RD) experiments are highly effective for probing micro- to millisecond conformational exchange processes in proteins. By performing experiments at multiple magnetic field strengths , one can extract dynamic parameters such as exchange rates, population fractions, and chemical shift differences. PySyncDyn is a comprehensive Python-based toolkit that automates the entire workflow from raw data processing to the generation of Dynamic Correlation (SyncDyn) maps. The workflow includes the calculation of effective transverse relaxation rates , pairwise fitting using the Carver-Richards model, generation of correlation maps, and computation of a SyncDyn Score that quantifies the extent of correlated dynamics across the protein. In addition, the Score2Pymol.py script allows visualization of these scores on the three-dimensional structure of the protein in PyMOL.
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    Domain insertion scanning to study and engineer ion channels
    (2021-01) Coyote-Maestas, Willow
    New proteins primarily evolve through recombining modular protein domains with discrete structure and function. Often domain recombination combines a catalytic functioning domain with a sensing domain, so protein function can be regulated by different stimuli. This form of domain recombination-based evolution underlies the intricate signaling networks that allow our cells and by extension our bodies to sense and respond to stimuli. Protein engineers mimic nature by combining domains with desirable properties into new useful combinations never seen in nature. This approach for generating synthetic multi-domain proteins has yielded groundbreaking therapeutics and tools for biology, most notably Car-T cancer therapies and GCaMP calcium sensors. However, domain recombination is challenging and requiring years of iterative optimization but unlike evolution we don’t have millions of years to spare. Both, our basic understanding of the biophysical principles of how proteins evolve and how to better engineer proteins are limited by a lack of domain compatibility rules. In the work presented in this thesis, we sought out to apply massively parallel domain insertion experiments and learn rules for domain compatibility. As our target protein, we used ion channels as they are an attractive engineering target and ion channels evolved through extensive domain recombination. Initially we started with a small set of 3 inserted domains inserted into all amino acid positions of a potassium channel kir2.1. We successfully engineered a light-switchable potassium channel that could be used by neuroscientists, however we found a tremendous amount of variability that necessitated expanding out to a broader sample of domain recombination space. Before we could achieve this goal, we needed to improve experimental pipelines because the methods the domain insertion field used at the time were not scalable nor generalizable. We developed a new domain insertion library generation method, SPINE, that yielded near perfect libraries. SPINE allowed us to expand out to over 700 different inserted domains with which we exhaustively sampled insertional space and developed a mechanistic model of domain recombination. We then expanded outward to several additional recipient channels to benchmark our work. Overall, we made major strides towards the goal of a mechanistic model for assembling protein domains. We expect this body of work will provide a foundation that will make domain-based engineering more effective and improve our understanding of the fundamentals of how proteins evolve.
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    On The Allosteric Mechanisms Of Paradoxical Activation By Raf Inhibitors
    (2024-04) Rasmussen, Damien
    The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.
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    On the Role of Allosteric Cooperativity in the Regulation of Protein Kinase A and its Implications in Disease
    (2021-05) Walker, Caitlin
    First articulated half a century ago, allostery has remained a universal phenomenon and is essential in understanding processes beyond the molecular level, such as cellular signaling and disease. Allostery also referred to as allosteric regulation, is a process by which biological macromolecules transmit the effect of binding at one site to an often distal, functional site, allowing for regulation. To facilitate the modulation of function between sites, allosteric signal is propagated through conserved amino acid residues, often comprising various structural elements of a protein. In general, allosteric communication is of fundamental interest and potentially of high relevance for drug design and protein engineering. Furthermore, the dysfunction of allosteric networks has been implicated in the etiology of human diseases. However, defining these networks of residues that mediate crosstalk between distal sites remains experimentally challenging and thus, poorly characterized. Since allosteric signal propagation relies on subtle conformational rearrangements, nuclear magnetic resonance (NMR) has emerged as an instrumental tool in investigating allosteric communication. This thesis aims to map allosteric networks at atomic resolution to understand how mutations in protein kinase A (PKA) influence allosteric communication to elicit the progression of various disease states. In this work we demonstrate how disease mutations associated with Cushing’s Syndrome and Fibrolamellar Hepatocellular Carcinoma attenuate the allosteric network of PKA, thereby disrupting the finely tuned regulation, specificity, and activation of PKA to generate dysfunctional signaling. The findings of this thesis provide critical insights into the importance of intramolecular allostery in facilitating functional signaling, directly showing how changes in allosteric networks of proteins lead to dysfunction.
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    Towards the full molecular investigation of protein kinase a mediated catalysis by NMR spectroscopy.
    (2009-03) Masterson, Larry Raou
    The reversible phosphorylation of proteins is fundamental to the modulation of myocardial contraction. A mechanism which controls this modulation occurs through alterations of Ca2+ flux formed across the sarcoplasmic reticulum (SR) membrane inside cardiomyocytes. Changes in this flux have a profound dependence on the interactions of three proteins: protein kinase A (PKA), sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), and phospholamban (PLN). Phosphorylation of PLN by PKA is associated with an augmented rate of SR Ca2+ uptake and relaxation of the myocardium. Mutants of PLN (R9C-PLN and R14Del-PLN) have previously been shown to be linked with forms of the fatal hereditary disease, dilated cardiomyopathy. The molecular basis of disease in this situation could result from irregularities in the association of these PLN mutants with PKA. The work presented here lays the foundation for obtaining the molecular details which govern these interactions to further our understanding of the processes which control Ca2+ transport in myocytes and, perhaps, lend insight into the origins of this disease.