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Open Access Publications from the University of California

Scholarly Works (18 results)

  • Article
  • Peer Reviewed
UC Berkeley Previously Published Works (2019)
The functionally tolerated sequence space of proteins can now be explored in an unprecedented way, owing to the expansion of genomic databases and the development of high-throughput methods to interrogate protein function. For signaling proteins, several recent studies have shown how the analysis of sequence variation leverages the available protein-structure information to provide new insights into specificity and allosteric regulation. In this Review, we discuss recent work that illustrates how this emerging approach is providing a deeper understanding of signaling proteins.
    Cover page: Understanding molecular mechanisms in cell signaling through natural and artificial sequence variation
    • Thesis
    • Peer Reviewed
    UC Berkeley Electronic Theses and Dissertations (2023)

    There is a growing need for improvements in renewable energy sources and in energy storage devices as the effects of global warming become more acute. Conventional lithium-ion batteries are composed of a lithium-graphite composite anode, a liquid electrolyte and a transition metal oxide cathode. Replacing the lithium-graphite anode with a lithium metal anode would greatly increase the energy density of these batteries, enabling higher range electric vehicles and significant improvements in consumer electronics. However, lithium metal anodes are incompatible with conventional liquid electrolytes, prone to dendrites and pose significant safety hazards. There has been significant research into replacing conventional liquid electrolytes with polymer electrolytes, which are significantly less flammable than liquid electrolytes, and have a higher modulus thereby suppressing dendrite growth. However, current polymer electrolytes cannot match the ion transport characteristics of conventional liquid electrolytes. To address this, researchers have attempted to combine various polymeric components with lithium salt to create an electrolyte that is both highly conductive and mechanically rigid. The thermodynamics of conventional polymer electrolytes are still poorly understood. In this Dissertation we study the effect of added salt on the thermodynamic properties of block copolymers and polymer blends comprised of poly(ethylene oxide) (PEO) and poly(methylmethacrylate) (PMMA). This Dissertation represents the first comprehensive study of the thermodynamics of a miscible polymer electrolyte system.

    In Chapter 2, we synthesize a series of PEO-PMMA block copolymers and analyze the effect of added lithium bis(trifluoromethane) sulfonimide (LiTFSI) salt on the phase behavior utilizing small angle X-ray scattering (SAXS). We calculate thermodynamics interaction parameters for this system and find that the effective thermodynamic interaction parameter, χeff, varies nonmonotonically with respect to salt concentration. We shed light upon the complex phase separation of PEO-PMMA/LiTFSI block copolymer electrolytes, which deviates from conventional block copolymer electrolytes.

    In Chapter 3, we prepare a series of PEO/PMMA/LiTFSI blend electrolytes and analyze the phase behavior of these blends via small angle neutron scattering. We find that both blend composition and salt concentration have a significant effect on polymer blend electrolyte phase behavior. We extract thermodynamic interaction parameters from the collected scattering data and build a thermodynamic model to predict blend phase behavior. We find that our model is in good agreement with our experimental data.

    In Chapter 4, we expand on our characterization of PEO/PMMA/LiTFSI blend electrolyte phase behavior by using light scattering to augment our previous phase characterization work. We create a comprehensive phase diagram of PEO/PMMA/LiTFSI polymer blends. This phase diagram presents some of the first experimental evidence of multiple immiscible windows in polymer blend electrolytes. We utilize our previously developed thermodynamic model to create a simulated phase diagram and find good agreement between theory and experiment.

    This work provides new insights into polymer-salt interactions and the underlying thermodynamics of polymer electrolytes. The goal of this Dissertation is to further analyze the complex thermodynamics of polymer electrolytes to enable design of future polymer electrolytes for lithium metal batteries.

      Cover page: Thermodynamics of Miscible Polymer Electrolytes
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2018)
      Upon activation, the epidermal growth factor receptor (EGFR) phosphorylates tyrosine residues in its cytoplasmic tail, which triggers the binding of Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains and initiates downstream signaling. The sequences flanking the tyrosine residues (referred to as "phosphosites") must be compatible with phosphorylation by the EGFR kinase domain and the recruitment of adapter proteins, while minimizing phosphorylation that would reduce the fidelity of signal transmission. To understand how phosphosite sequences encode these functions within a small set of residues, we carried out high-throughput mutational analysis of three phosphosite sequences in the EGFR tail. We used bacterial surface display of peptides coupled with deep sequencing to monitor phosphorylation efficiency and the binding of the SH2 and PTB domains of the adapter proteins Grb2 and Shc1, respectively. We found that the sequences of phosphosites in the EGFR tail are restricted to a subset of the range of sequences that can be phosphorylated efficiently by EGFR. Although efficient phosphorylation by EGFR can occur with either acidic or large hydrophobic residues at the -1 position with respect to the tyrosine, hydrophobic residues are generally excluded from this position in tail sequences. The mutational data suggest that this restriction results in weaker binding to adapter proteins but also disfavors phosphorylation by the cytoplasmic tyrosine kinases c-Src and c-Abl. Our results show how EGFR-family phosphosites achieve a trade-off between minimizing off-pathway phosphorylation and maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2018)
        The specificity of tyrosine kinases is attributed predominantly to localization effects dictated by non-catalytic domains. We developed a method to profile the specificities of tyrosine kinases by combining bacterial surface-display of peptide libraries with next-generation sequencing. Using this, we showed that the tyrosine kinase ZAP-70, which is critical for T cell signaling, discriminates substrates through an electrostatic selection mechanism encoded within its catalytic domain (Shah et al., 2016). Here, we expand this high-throughput platform to analyze the intrinsic specificity of any tyrosine kinase domain against thousands of peptides derived from human tyrosine phosphorylation sites. Using this approach, we find a difference in the electrostatic recognition of substrates between the closely related Src-family kinases Lck and c-Src. This divergence likely reflects the specialization of Lck to act in concert with ZAP-70 in T cell signaling. These results point to the importance of direct recognition at the kinase active site in fine-tuning specificity.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2022)
          We investigate a polyacetal-based electrolyte, poly(1,3,6-Trioxocane) (P(2EO-MO)) mixed with lithium bis(trifluoromethanesulfonyl) imide (LiTFSI) salt, and report full electrochemical characterization of the transport parameters and a thermodynamic property in comparison to the previously reported poly(ethylene oxide) (PEO) electrolyte data [D. Gribble et al., J. Electrochem. Soc., 166, A3228 (2019)]. While the steady-state current fraction (?+) of P(2EO-MO) electrolyte is greater than that of PEO electrolyte in the entire salt concentration window we explored, the rigorously defined transference number using Newman s concentrated solution theory ( + t 0) appears to be similar to that of PEO electrolyte. On the basis of full electrochemical characterization, we calculate the salt concentration profile as a function of position in the cell and predict limiting current density (iLL) as a function of salt concentration. Experimental data were compared to the predicted values. The non-monotonic behaviors were observed both in prediction and experimental results with offset peak positions. We find that the limiting current density of P (2EO-MO) electrolyte is systematically lower than that of PEO electrolyte in most of the salt concentrations with the exception of rav = 0.05. It is noteworthy that even though one measure of electrolyte efficacy (?+) is superior in P(2EO-MO) electrolyte, the limiting current density, which is another metric of electrolyte efficacy at high currents, is not greater in P(2EO-MO).
            Cover page: Complete Electrochemical Characterization and Limiting Current of Polyacetal Electrolytes
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2021)
            We studied the effect of salt addition on a diblock copolymer system with a negative Flory-Huggins interaction parameter, χ, indicative of attractive interactions between the two blocks. The system studied is poly(ethylene oxide)-block-poly(methyl methacrylate) (PEO-PMMA) with added lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) salt. We studied two asymmetric block copolymers, PEO-PMMA(10-33) and PEO-PMMA(10-64), where the numbers refer to the molar masses of the blocks in kg mol-1. The small-angle X-ray scattering (SAXS) profiles for PEO-PMMA(10-33) were featureless at all salt concentrations. In contrast, PEO-PMMA(10-64) exhibited SAXS peaks when the salt concentration was between 0.22 ≤ m (mol Li/kg polymer) ≤ 0.44. The appearance of SAXS peaks only in PEO-PMMA(10-64) is consistent with the predictions of ionic self-consistent field theory developed by de la Cruz and co-workers, which predicts that in systems with negative χ, ordered phases are only found when the volume fraction of the ionic block is about 10%.
              Cover page: Effect of Added Salt on Disordered Poly(ethylene oxide)-Block-Poly(methyl methacrylate) Copolymer Electrolytes
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2018)
              T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling.
                Cover page: Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2016)
                The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens.
                  Cover page: An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2019)
                  Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.
                    Cover page: Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination
                    • Article
                    • Peer Reviewed
                    UC Berkeley Previously Published Works (2017)
                    Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins.
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