Allan Chris M. Ferreon, Josephine C. Ferreon, Peter E. Wright, and Ashok A. Deniz
Allostery is an intrinsic property of many globular proteins and enzymes that is indispensable for cellular regulatory and feedback mechanisms. Recent theoretical and empirical observations indicate that allostery is also manifest in intrinsically disordered proteins, which account for a substantial proportion of the proteome. Many intrinsically disordered proteins are promiscuous binders that interact with multiple partners and frequently function as molecular hubs in protein interaction networks. The adenovirus early region 1A (E1A) oncoprotein is a prime example of a molecular hub intrinsically disordered protein. E1A can induce marked epigenetic reprogramming of the cell within hours after infection, through interactions with a diverse set of partners that include key host regulators such as the general transcriptional coactivator CREB binding protein (CBP), its paralogue p300, and the retinoblastoma protein (pRb; also called RB1). Little is known about the allosteric effects at play in E1A–CBP–pRb interactions, or more generally in hub intrinsically disordered protein interaction networks. Here we used single-molecule fluorescence resonance energy transfer (smFRET) to study coupled binding and folding processes in the ternary E1A system. The low concentrations used in these high-sensitivity experiments proved to be essential for these studies, which are challenging owing to a combination of E1A aggregation propensity and high-affinity binding interactions. Our data revealed that E1A–CBP–pRb interactions have either positive or negative cooperativity, depending on the available E1A interaction sites. This striking cooperativity switch enables fine-tuning of the thermodynamic accessibility of the ternary versus binary E1A complexes, and may permit a context-specific tuning of associated downstream signalling outputs. Such a modulation of allosteric interactions is probably a common mechanism in molecular hub intrinsically disordered protein function.
DOI
Journal: Nature
Ionel Popa, Pallav Kosuri, Jorge Alegre-Cebollada, Sergi Garcia-Manyes, and Julio M Fernandez
Here we describe a protocol for using force-clamp spectroscopy to precisely quantify the effect of force on biochemical reactions. A calibrated force is used to control the exposure of reactive sites in a single polyprotein substrate composed of repeated domains. The use of polyproteins allows the identification of successful single-molecule recordings from unambiguous mechanical unfolding fingerprints. Biochemical reactions are then measured directly by detecting the length changes of the substrate held at a constant force. We present the layout of a force-clamp spectrometer along with protocols to design and conduct experiments. These experiments measure reaction kinetics as a function of applied force. We show sample data of the force dependency of two different reactions, protein unfolding and disulfide reduction. These data, which can be acquired in just a few days, reveal mechanistic details of the reactions that currently cannot be resolved by any other technique.
DOI
Journal: Nature Protocols
Deep Punj, Mathieu Mivelle, Satish Babu Moparthi, Thomas S. van Zanten, Hervé Rigneault, Niek F. van Hulst, María F. García-Parajó, and Jérôme Wenger
Single-molecule fluorescence techniques are key for a number of applications, including DNA sequencing, molecular and cell biology and early diagnosis. Unfortunately, observation of single molecules by diffraction-limited optics is restricted to detection volumes in the femtolitre range and requires pico- or nanomolar concentrations, far below the micromolar range where most biological reactions occur. This limitation can be overcome using plasmonic nanostructures, which enable the confinement of light down to nanoscale volumes. Although these nanoantennas enhance fluorescence brightness, large background signals and/or unspecific binding to the metallic surface have hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel ‘antenna-in-box’ platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single-molecule sensitivity (fluorescence enhancement up to 1,100-fold and microsecond transit times) at micromolar sample concentrations and zeptolitre-range detection volumes. The antenna-in-box device can be optimized for single-molecule fluorescence studies at physiologically relevant concentrations, as we demonstrate using various biomolecules.
DOI
Journal: Nature Nanotechnology
Ruifeng Qi, Evans Boateng Sarbeng, Qun Liu, Katherine Quynh Le, Xinping Xu, Hongya Xu, Jiao Yang, Jennifer Li Wong, Christina Vorvis, Wayne A Hendrickson, Lei Zhou, and Qinglian Liu
The 70-kilodalton (kDa) heat-shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD), which binds and hydrolyzes ATP, and a substrate-binding domain (SBD), which binds extended polypeptides. NBD and SBD interact little when in the presence of ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact ATP-bound Hsp70 from Escherichia coli at 1.96-Å resolution. The ATP-bound NBD adopts a unique conformation, forming extensive interfaces with an SBD that has changed radically, having its α-helical lid displaced and the polypeptide-binding channel of its β-subdomain restructured. These conformational changes, together with our biochemical assays, provide a structural explanation for allosteric coupling in Hsp70 activity.
DOI
Journal:Nature Structural & Molecular Biology
Xuefeng Wang, Taekjip Ha
Cell-cell and cell-matrix mechanical interactions through membrane receptors direct a wide range of cellular functions and orchestrate the development of multicellular organisms. To define the single molecular forces required to activate signaling through a ligand-receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor-ligand tension. Using a range of tethers with tunable tension tolerances, we show that cells apply a universal peak tension of about 40 piconewtons (pN) to single integrin-ligand bonds during initial adhesion. We find that less than 12 pN is required to activate Notch receptors. TGT can also provide a defined molecular mechanical cue to regulate cellular functions.
DOI
Journal: Science
Gabriel Villar, Alexander D. Graham, and Hagan Bayley
Living cells communicate and cooperate to produce the emergent properties of tissues. Synthetic mimics of cells, such as liposomes, are typically incapable of cooperation and therefore cannot readily display sophisticated collective behavior. We printed tens of thousands of picoliter aqueous droplets that become joined by single lipid bilayers to form a cohesive material with cooperating compartments. Three-dimensional structures can be built with heterologous droplets in software-defined arrangements. The droplet networks can be functionalized with membrane proteins; for example, to allow rapid electrical communication along a specific path. The networks can also be programmed by osmolarity gradients to fold into otherwise unattainable designed structures. Printed droplet networks might be interfaced with tissues, used as tissue engineering substrates, or developed as mimics of living tissue.
DOI
Journal: Science
Mian Zhou, Jinhu Guo, Joonseok Cha, Michael Chae, She Chen, Jose M. Barral, Matthew S. Sachs, and Yi Liu
Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes1, 2, 3. Codon usage is also implicated in the control of transcription, splicing and RNA structure4, 5, 6. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.
DOI
Journal: Nature
Allostery is an intrinsic property of many globular proteins and enzymes that is indispensable for cellular regulatory and feedback mechanisms. Recent theoretical and empirical observations indicate that allostery is also manifest in intrinsically disordered proteins, which account for a substantial proportion of the proteome. Many intrinsically disordered proteins are promiscuous binders that interact with multiple partners and frequently function as molecular hubs in protein interaction networks. The adenovirus early region 1A (E1A) oncoprotein is a prime example of a molecular hub intrinsically disordered protein. E1A can induce marked epigenetic reprogramming of the cell within hours after infection, through interactions with a diverse set of partners that include key host regulators such as the general transcriptional coactivator CREB binding protein (CBP), its paralogue p300, and the retinoblastoma protein (pRb; also called RB1). Little is known about the allosteric effects at play in E1A–CBP–pRb interactions, or more generally in hub intrinsically disordered protein interaction networks. Here we used single-molecule fluorescence resonance energy transfer (smFRET) to study coupled binding and folding processes in the ternary E1A system. The low concentrations used in these high-sensitivity experiments proved to be essential for these studies, which are challenging owing to a combination of E1A aggregation propensity and high-affinity binding interactions. Our data revealed that E1A–CBP–pRb interactions have either positive or negative cooperativity, depending on the available E1A interaction sites. This striking cooperativity switch enables fine-tuning of the thermodynamic accessibility of the ternary versus binary E1A complexes, and may permit a context-specific tuning of associated downstream signalling outputs. Such a modulation of allosteric interactions is probably a common mechanism in molecular hub intrinsically disordered protein function.
