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"Shah, Neel"
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البرمجة اللغوية العصبية NLP
هذا الكتاب هو مقدمة عملية وممتعة للبرمجة اللغوية العصبية وتقنيات التعلم المتسارعة المكتوبة بأسلوب يتيح لك تطبيق ما تعلمته فورا عن مواقف العالم الحقيقي حيث وصفت البرمجة اللغوية العصبية بأنها المنهج النفي الأهم الذي يمكن الناس من الحصول على حياة أفضل وأكمل أصبح عالم البرمجة اللغوية العصبية للأسف مليئا بالمصطلحات والتعابر التقنية الطنانة التي تقيد الناس وتمنعهم في الحقيقة من الاستفادة من المناهج البسيطة التي يمكن أن تحدث تأثرا عميقا عن حياتهم.
BOOK
Understanding molecular mechanisms in cell signaling through natural and artificial sequence variation
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.
Journal Article
Nanosecond solvation dynamics in a polymer electrolyte for lithium batteries
Solvation dynamics critically affect charge transport. Spectroscopic experiments and computer simulations show that these dynamics in aqueous systems occur on a picosecond timescale. In the case of organic electrolytes, however, conflicting values ranging from 1 to several 100 picoseconds have been reported. We resolve this conflict by studying mixtures of an organic polymer and a lithium salt. Lithium ions coordinate with multiple polymer chains, resulting in temporary crosslinks. Relaxation of these crosslinks, detected by quasielastic neutron scattering, are directly related to solvation dynamics. Simulations reveal a broad spectrum of relaxation times. The average timescale for solvation dynamics in both experiment and simulation is one nanosecond. We present the direct measurement of ultraslow dynamics of solvation shell break-up in an electrolyte.
Solvation dynamics at picosecond timescales critically affect charge transport in aqueous systems, but conflicting values have been reported for organic electrolytes. Lifetimes on the order of 1 ns for mixtures of organic polymer and lithium salt exhibiting ultraslow dynamics of solvation shell break-up are now reported.
Journal Article
A promiscuous split intein with expanded protein engineering applications
The protein trans-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an “extein dependence,” whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided protein engineering approach that imbues ultrafast DnaE split inteins with minimal extein dependence. The resulting “promiscuous” inteins are shown to be superior reagents for protein cyclization and protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins.
Journal Article
Deep mutational scanning of the multi-domain phosphatase SHP2 reveals mechanisms of regulation and pathogenicity
Multi-domain signaling enzymes are often regulated through extensive inter-domain interactions, and disruption of inter-domain interfaces by mutations can lead to aberrant signaling and diseases. For example, the tyrosine phosphatase SHP2 contains two phosphotyrosine recognition domains that auto-inhibit its catalytic domain. SHP2 is canonically activated by binding of these non-catalytic domains to phosphoproteins, which destabilizes its auto-inhibited state, and several mutations at the main auto-inhibitory interface have been shown to hyperactivate SHP2 in cancers and developmental disorders. Hundreds of clinically observed mutations in SHP2 have not been characterized, but their locations suggest alternative modes of dysregulation. Here, we perform deep mutational scanning on full-length SHP2 and the isolated phosphatase domain to dissect mechanisms of dysregulation. Our analysis reveals mechanistically diverse mutational effects and identifies key intra- and inter-domain interactions that contribute to SHP2 activity, dynamics, and regulation. Our datasets also provide insights into the potential pathogenicity of previously uncharacterized clinical variants.
Comprehensive characterization of mutational effects in the human phosphatase SHP2 reveals mechanisms of intra- and inter-domain regulation, shedding light on the mechanisms of pathogenicity for hundreds of disease-associated mutations.
Journal Article
Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT
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.
TCR signaling initiates a signaling cascade involving the kinases Lck and Zap70 and the adaptor LAT. Weiss and colleagues discover a proline-rich motif in LAT, which facilitates interactions among Lck, LAT and Zap70 for efficient TCR signaling.
Journal Article
Prediction of protein–ligand binding affinity from sequencing data with interpretable machine learning
Protein–ligand interactions are increasingly profiled at high throughput using affinity selection and massively parallel sequencing. However, these assays do not provide the biophysical parameters that most rigorously quantify molecular interactions. Here we describe a flexible machine learning method, called ProBound, that accurately defines sequence recognition in terms of equilibrium binding constants or kinetic rates. This is achieved using a multi-layered maximum-likelihood framework that models both the molecular interactions and the data generation process. We show that ProBound quantifies transcription factor (TF) behavior with models that predict binding affinity over a range exceeding that of previous resources; captures the impact of DNA modifications and conformational flexibility of multi-TF complexes; and infers specificity directly from in vivo data such as ChIP-seq without peak calling. When coupled with an assay called
K
D
-seq, it determines the absolute affinity of protein–ligand interactions. We also apply ProBound to profile the kinetics of kinase–substrate interactions. ProBound opens new avenues for decoding biological networks and rationally engineering protein–ligand interactions.
Protein–ligand binding affinity is predicted quantitatively from sequencing data.
Journal Article
Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites
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.
Journal Article
High-throughput profiling of sequence recognition by tyrosine kinases and SH2 domains using bacterial peptide display
Tyrosine kinases and SH2 (phosphotyrosine recognition) domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue. Although the preferred recognition motifs of many kinases and SH2 domains are known, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases and SH2 domains. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences. We also screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. We extended this platform to the analysis of SH2 domains and showed that screens could predict relative binding affinities. Finally, we expanded our method to assess the impact of non-canonical and post-translationally modified amino acids on sequence recognition. This specificity profiling platform will shed new light on phosphotyrosine signaling and could readily be adapted to other protein modification/recognition domains.
Journal Article
Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen
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.
Journal Article