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1.
Nat Commun ; 14(1): 2875, 2023 05 19.
Article in English | MEDLINE | ID: mdl-37208363

ABSTRACT

Engineering protein biosensors that sensitively respond to specific biomolecules by triggering precise cellular responses is a major goal of diagnostics and synthetic cell biology. Previous biosensor designs have largely relied on binding structurally well-defined molecules. In contrast, approaches that couple the sensing of flexible compounds to intended cellular responses would greatly expand potential biosensor applications. Here, to address these challenges, we develop a computational strategy for designing signaling complexes between conformationally dynamic proteins and peptides. To demonstrate the power of the approach, we create ultrasensitive chemotactic receptor-peptide pairs capable of eliciting potent signaling responses and strong chemotaxis in primary human T cells. Unlike traditional approaches that engineer static binding complexes, our dynamic structure design strategy optimizes contacts with multiple binding and allosteric sites accessible through dynamic conformational ensembles to achieve strongly enhanced signaling efficacy and potency. Our study suggests that a conformationally adaptable binding interface coupled to a robust allosteric transmission region is a key evolutionary determinant of peptidergic GPCR signaling systems. The approach lays a foundation for designing peptide-sensing receptors and signaling peptide ligands for basic and therapeutic applications.


Subject(s)
Chemotaxis , Peptides , Humans , Chemotaxis/physiology , Signal Transduction , Proteins , Allosteric Site , Ligands
2.
Nat Commun ; 13(1): 6826, 2022 11 11.
Article in English | MEDLINE | ID: mdl-36369272

ABSTRACT

Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can be readily characterized, receptor associations into quaternary structures are challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed binding interactions, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers activated the G protein Gi, but only specific dimer structures also recruited ß-arrestins. Overall, we demonstrate that quaternary structures represent an important unforeseen mechanism of receptor biased signaling and reveal the existence of a bias switch at the dimer interface of several G protein-coupled receptors including CXCR4, mu-Opioid and type-2 Vasopressin receptors that selectively control the activation of G proteins vs ß-arrestin-mediated pathways. The approach should prove useful for predicting and designing receptor associations to uncover and reprogram selective cellular signaling functions.


Subject(s)
Arrestins , Signal Transduction , Arrestins/metabolism , beta-Arrestins/metabolism , Signal Transduction/physiology , Receptors, G-Protein-Coupled/metabolism , GTP-Binding Proteins/metabolism
3.
Nat Chem Biol ; 14(5): 489-496, 2018 05.
Article in English | MEDLINE | ID: mdl-29581582

ABSTRACT

ClC chloride channels and transporters are important for chloride homeostasis in species from bacteria to human. Mutations in ClC proteins cause genetically inherited diseases, some of which are likely to involve folding defects. The ClC proteins present a challenging and unusual biological folding problem because they are large membrane proteins possessing a complex architecture, with many reentrant helices that go only partway through membrane and loop back out. Here we were able to examine the unfolding of the Escherichia coli ClC transporter, ClC-ec1, using single-molecule forced unfolding methods. We found that the protein could be separated into two stable halves that unfolded independently. The independence of the two domains is consistent with an evolutionary model in which the two halves arose from independently folding subunits that later fused together. Maintaining smaller folding domains of lesser complexity within large membrane proteins may be an advantageous strategy to avoid misfolding traps.


Subject(s)
Chloride Channels/chemistry , Chlorides/chemistry , Escherichia coli/chemistry , DNA/chemistry , Dimyristoylphosphatidylcholine/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Evolution, Molecular , Humans , Membrane Transport Proteins/chemistry , Molecular Dynamics Simulation , Mutation , Plasmids , Protein Denaturation , Protein Domains , Protein Folding , Protein Multimerization , Protein Structure, Secondary
4.
J Mol Biol ; 430(4): 424-437, 2018 02 16.
Article in English | MEDLINE | ID: mdl-28549924

ABSTRACT

Protein folding is a fundamental life process with many implications throughout biology and medicine. Consequently, there have been enormous efforts to understand how proteins fold. Almost all of this effort has focused on water-soluble proteins, however, leaving membrane proteins largely wandering in the wilderness. The neglect has occurred not because membrane proteins are unimportant but rather because they present many theoretical and technical complications. Indeed, quantitative membrane protein folding studies are generally restricted to a handful of well-behaved proteins. Single-molecule methods may greatly alter this picture, however, because the ability to work at or near infinite dilution removes aggregation problems, one of the main technical challenges of membrane protein folding studies.


Subject(s)
Fluorescence Resonance Energy Transfer/methods , Mass Spectrometry/methods , Membrane Proteins/chemistry , Microscopy, Atomic Force/methods , Protein Folding , Single Molecule Imaging/methods , Animals , Humans , Membrane Lipids/chemistry , Membrane Proteins/isolation & purification
5.
Protein Sci ; 25(8): 1535-44, 2016 08.
Article in English | MEDLINE | ID: mdl-27222403

ABSTRACT

Manipulating single molecules and systems of molecules with mechanical force is a powerful technique to examine their physical properties. Applying force requires attachment of the target molecule to larger objects using some sort of molecular tether, such as a strand of DNA. DNA handle attachment often requires difficult manipulations of the target molecule, which can preclude attachment to unstable, hard to obtain, and/or large, complex targets. Here we describe a method for covalent DNA handle attachment to proteins that simply requires the addition of a preprepared reagent to the protein and a short incubation. The handle attachment method developed here provides a facile approach for studying the biomechanics of biological systems.


Subject(s)
DNA-Binding Proteins/chemistry , DNA/chemistry , Endopeptidases/chemistry , Escherichia coli Proteins/chemistry , Maltose-Binding Proteins/chemistry , Membrane Proteins/chemistry , Protein Engineering/methods , Recombinant Fusion Proteins/chemistry , Staining and Labeling/methods , Amino Acid Sequence , Biomechanical Phenomena , Cloning, Molecular , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimyristoylphosphatidylcholine/chemistry , Endopeptidases/genetics , Endopeptidases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression , Lipid Bilayers/chemistry , Magnets , Maltose-Binding Proteins/genetics , Maltose-Binding Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Optical Tweezers , Peptides/chemical synthesis , Peptides/chemistry , Peptides/metabolism , Protein Folding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
6.
Nat Chem Biol ; 11(12): 981-7, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26479439

ABSTRACT

Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single-molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, Escherichia coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (ΔG = 6.5 kBT) but a large unfolding barrier (21.3 kBT) that can maintain the protein in a folded state for long periods of time (t1/2 ∼3.5 h). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure.


Subject(s)
Escherichia coli Proteins/chemistry , Membrane Proteins/chemistry , Thermodynamics , Escherichia coli Proteins/metabolism , Kinetics , Membrane Proteins/metabolism , Models, Molecular , Protein Conformation , Protein Folding
7.
J Am Chem Soc ; 135(40): 15183-90, 2013 Oct 09.
Article in English | MEDLINE | ID: mdl-24032628

ABSTRACT

Approximately 10% of water-soluble proteins are considered kinetically stable with unfolding half-lives in the range of weeks to millenia. These proteins only rarely sample the unfolded state and may never unfold on their respective biological time scales. It is still not known whether membrane proteins can be kinetically stable, however. Here we examine the subunit dissociation rate of the trimeric membrane enzyme, diacylglycerol kinase, from Escherichia coli as a proxy for complete unfolding. We find that dissociation occurs with a half-life of at least several weeks, demonstrating that membrane proteins can remain locked in a folded state for long periods of time. These results reveal that evolution can use kinetic stability to regulate the biological function of membrane proteins, as it can for soluble proteins. Moreover, it appears that the generation of kinetic stability could be a viable target for membrane protein engineering efforts.


Subject(s)
Diacylglycerol Kinase/chemistry , Diacylglycerol Kinase/metabolism , Enzyme Activation , Enzyme Stability , Escherichia coli/enzymology , Kinetics , Models, Molecular , Protein Multimerization , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Unfolding
8.
Proc Natl Acad Sci U S A ; 107(16): 7509-14, 2010 Apr 20.
Article in English | MEDLINE | ID: mdl-20308536

ABSTRACT

Hundreds of bacterial species produce proteinaceous microcompartments (MCPs) that act as simple organelles by confining the enzymes of metabolic pathways that have toxic or volatile intermediates. A fundamental unanswered question about bacterial MCPs is how enzymes are packaged within the protein shell that forms their outer surface. Here, we report that a short N-terminal peptide is necessary and sufficient for packaging enzymes into the lumen of an MCP involved in B(12)-dependent 1,2-propanediol utilization (Pdu MCP). Deletion of 10 or 14 amino acids from the N terminus of the propionaldehyde dehydrogenase (PduP) enzyme, which is normally found within the Pdu MCP, substantially impaired packaging, with minimal effects on its enzymatic activity. Fusion of the 18 N-terminal amino acids from PduP to GFP, GST, or maltose-binding protein resulted in their encapsulation within MCPs. Bioinformatic analyses revealed N-terminal extensions in two additional Pdu proteins and three proteins from two unrelated MCPs, suggesting that N-terminal peptides may be used to package proteins into diverse MCPs. The potential uses of MCP assembly principles in nature and in biotechnology are discussed.


Subject(s)
Bacteria/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Computational Biology/methods , Green Fluorescent Proteins/chemistry , Maltose-Binding Proteins , Microscopy, Fluorescence/methods , Models, Genetic , Molecular Sequence Data , Periplasmic Binding Proteins/chemistry , Propylene Glycol/chemistry , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Salmonella enterica/metabolism , Sequence Homology, Amino Acid , Vitamin B 12/metabolism
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