Journal of Molecular Biology

ScienceDirect RSS
  • Computational De Novo Design of a Self-Assembling Peptide with Predefined Structure
    [Jan 2015]

    Publication date: 30 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 2

    Author(s): Sabine Kaltofen , Chenge Li , Po-Ssu Huang , Louise C. Serpell , Andreas Barth , Ingemar André

    Protein and peptide self-assembly is a powerful design principle for engineering of new biomolecules. More sophisticated biomaterials could be built if both the structure of the overall assembly and that of the self-assembling building block could be controlled. To approach this problem, we developed a computational design protocol to enable de novo design of self-assembling peptides with predefined structure. The protocol was used to design a peptide building block with a βαβ fold that self-assembles into fibrillar structures. The peptide associates into a double β-sheet structure with tightly packed α-helices decorating the exterior of the fibrils. Using circular dichroism, Fourier transform infrared spectroscopy, electron microscopy and X-ray fiber diffraction, we demonstrate that the peptide adopts the designed conformation. The results demonstrate that computational protein design can be used to engineer protein and peptide assemblies with predefined three-dimensional structures, which can serve as scaffolds for the development of functional biomaterials. Rationally designed proteins and peptides could also be used to investigate the subtle energetic and entropic tradeoffs in natural self-assembly processes and the relation between assembly structure and assembly mechanism. We demonstrate that the de novo designed peptide self-assembles with a mechanism that is more complicated than expected, in a process where small changes in solution conditions can lead to significant differences in assembly properties and conformation. These results highlight that formation of structured protein/peptide assemblies is often dependent on the formation of weak but highly precise intermolecular interactions.
    Graphical abstract




    Categories: Journal Articles
  • A General Computational Approach for Repeat Protein Design
    [Jan 2015]

    Publication date: 30 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 2

    Author(s): Fabio Parmeggiani , Po-Ssu Huang , Sergey Vorobiev , Rong Xiao , Keunwan Park , Silvia Caprari , Min Su , Jayaraman Seetharaman , Lei Mao , Haleema Janjua , Gaetano T. Montelione , John Hunt , David Baker

    Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1Å root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds.
    Graphical abstract




    Categories: Journal Articles
  • An Improved Single-Chain Fab Platform for Efficient Display and Recombinant Expression
    [Jan 2015]

    Publication date: 30 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 2

    Author(s): James T. Koerber , Michael J. Hornsby , James A. Wells

    Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1–3mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (T m of 81°C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.
    Graphical abstract




    Categories: Journal Articles
  • Editorial Board
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1









    Categories: Journal Articles
  • Contents List
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1









    Categories: Journal Articles
  • Insights into the molecular foundations of electrical excitation
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Rachelle Gaudet , Benoit Roux , Daniel L. MinorJr







    Categories: Journal Articles
  • Bacterial Voltage-Gated Sodium Channels (BacNaVs) from the Soil, Sea, and Salt Lakes Enlighten Molecular Mechanisms of Electrical Signaling and Pharmacology in the Brain and Heart
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Jian Payandeh , Daniel L. Minor Jr.

    Voltage-gated sodium channels (NaVs) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60years, functional studies of NaVs have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNaVs) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic NaVs have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNaVs as templates for drug development efforts.
    Graphical abstract




    Categories: Journal Articles
  • Ryanodine Receptors: Allosteric Ion Channel Giants
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Filip Van Petegem

    The endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) form major intracellular Ca2+ stores. Ryanodine receptors (RyRs) are large tetrameric ion channels in the SR and ER membranes that can release Ca2+ upon triggering. With molecular masses exceeding 2.2MDa, they represent the pinnacle of ion channel complexity. RyRs have adopted long-range allosteric mechanisms, with pore opening resulting in conformational changes over 200Å away. Together with tens of protein and small molecule modulators, RyRs have adopted rich and complex regulatory mechanisms. Structurally related to inositol-1,4,5-trisphosphate receptors (IP3Rs), RyRs have been studied extensively using cryo-electron microscopy (cryo-EM). Along with more recent X-ray crystallographic analyses of individual domains, these have resulted in pseudo-atomic models. Over 500 mutations in RyRs have been linked to severe genetic disorders, which underscore their role in the contraction of cardiac and skeletal muscles. Most of these have been linked to gain-of-function phenotypes, resulting in premature or prolonged leak of Ca2+ in the cytosol. This review outlines our current knowledge on the structure of RyRs at high and low resolutions, their relationship to IP3Rs, an overview of the most commonly studied regulatory mechanisms, and models that relate disease-causing mutations to altered channel function.
    Graphical abstract




    Categories: Journal Articles
  • Architectural and Functional Similarities between Trimeric ATP-Gated P2X Receptors and Acid-Sensing Ion Channels
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Stephan Kellenberger , Thomas Grutter

    ATP-gated P2X receptors and acid-sensing ion channels are two distinct ligand-gated ion channels that assemble into trimers. They are involved in many important physiological functions such as pain sensation and are recognized as important therapeutic targets. They have unrelated primary structures and respond to different ligands (ATP and protons) and are thus considered as two different ion channels. As a consequence, comparisons of the biophysical properties and underlying mechanisms have only been rarely made between these two channels. However, the recent determination of their molecular structures by X-ray crystallography has revealed unexpected parallels in the architecture of the two pores, providing a basis for possible functional analogies. In this review, we analyze the structural and functional similarities that are shared by these trimeric ion channels, and we outline key unanswered questions that, if addressed experimentally, may help us to elucidate how two unrelated ion channels have adopted a similar fold of the pore.
    Graphical abstract




    Categories: Journal Articles
  • The Enigmatic Cytoplasmic Regions of KCNH Channels
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): João H. Morais-Cabral , Gail A. Robertson

    KCNH channels are expressed across a vast phylogenetic and evolutionary spectrum. In humans, they function in a wide range of tissues and serve as biomarkers and targets for diseases such as cancer and cardiac arrhythmias. These channels share a general architecture with other voltage-gated ion channels but are distinguished by the presence of an N-terminal PAS (Per-Arnt-Sim) domain and a C-terminal domain with homology to cyclic nucleotide binding domains (referred to as the CNBh domain). Cytosolic regions outside these domains show little conservation between KCNH families but are strongly conserved across species within a family, likely reflecting variability that confers specificity to individual channel types. PAS and CNBh domains participate in channel gating, but at least twice in evolutionary history, the PAS domain has been lost and it is omitted by alternate transcription to create a distinct channel subunit in one family. In this focused review, we present current knowledge of the structure and function of these cytosolic regions, discuss their evolution as modular domains and provide our perspective on the important questions moving forward.
    Graphical abstract




    Categories: Journal Articles
  • Structural and Functional Mechanisms of CRAC Channel Regulation
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Ann Hye-Ryong Shim , Leidamarie Tirado-Lee , Murali Prakriya

    In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca2+ influx through store-operated Ca2+-release-activated Ca2+ (CRAC) channels. The ensuing Ca2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. The physiological importance of CRAC channels for human health is underscored by studies indicating that mutations in CRAC channel genes produce a spectrum of devastating diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Moreover, from a basic science perspective, CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca2+ selectivity, store-operated gating, and distinct pore properties and therefore serve as fascinating model ion channels for understanding the biophysical mechanisms of Ca2+ selectivity and channel opening. Studies in the last two decades have revealed the cellular and molecular choreography of the CRAC channel activation process, and it is now established that opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins and the endoplasmic reticulum Ca2+ sensors STIM1 and STIM2. In this review, we summarize the functional and structural mechanisms of CRAC channel regulation, focusing on recent advances in our understanding of the conformational and structural dynamics of CRAC channel gating.
    Graphical abstract




    Categories: Journal Articles
  • TMEM16 Proteins: Unknown Structure and Confusing Functions
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Alessandra Picollo , Mattia Malvezzi , Alessio Accardi

    The TMEM16 family of membrane proteins, also known as anoctamins, plays key roles in a variety of physiological functions that range from ion transport to phospholipid scrambling and to regulating other ion channels. The first two family members to be functionally characterized, TMEM16A (ANO1) and TMEM16B (ANO2), form Ca2+-activated Cl− channels and are important for transepithelial ion transport, olfaction, phototransduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. The roles of other family members, such as TMEM16C (ANO3), TMEM16D (ANO4), TMEM16F (ANO6), TMEM16G (ANO7) and TMEM16J (ANO9), remain poorly understood and controversial. These homologues were reported to be phospholipid scramblases, ion channels, to have both functions or to be regulatory subunits of other channels. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused by impaired Ca2+-dependent externalization of phosphatidylserine in activated platelets, suggesting that this homologue might be a scramblase. However, overexpression of TMEM16F has also been associated with a remarkable number of different ion channel types, raising the possibility that this protein might be involved in both ion and lipid transports. The recent identification of an ancestral TMEM16 homologue with intrinsic channel and scramblase activities supports this hypothesis. Thus, the TMEM16 family might have diverged in two or three different subclasses, channels, scramblases and dual-function channel/scramblases. The structural bases and functional implication of such a functional diversity within a single protein family remain to be elucidated and the links between TMEM16 functions and human physiology and pathologies need to be investigated.
    Graphical abstract




    Categories: Journal Articles
  • Restoration of NBD1 Thermal Stability Is Necessary and Sufficient to Correct ∆F508 CFTR Folding and Assembly
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Lihua He , Andrei A. Aleksandrov , Jianli An , Liying Cui , Zhengrong Yang , Christie G. Brouillette , John R. Riordan

    Cystic fibrosis transmembrane conductance regulator (CFTR) (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis. The most common cystic-fibrosis-causing mutation, the deletion of F508 (ΔF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The ΔF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question, we varied the extent of stabilization of NBD1 using different second-site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on ∆F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the ΔF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.
    Graphical abstract




    Categories: Journal Articles
  • Hydrophobic Gating in Ion Channels
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Prafulla Aryal , Mark S.P. Sansom , Stephen J. Tucker

    Biological ion channels are nanoscale transmembrane pores. When water and ions are enclosed within the narrow confines of a sub-nanometer hydrophobic pore, they exhibit behavior not evident from macroscopic descriptions. At this nanoscopic level, the unfavorable interaction between the lining of a hydrophobic pore and water may lead to stochastic liquid–vapor transitions. These transient vapor states are “dewetted”, i.e. effectively devoid of water molecules within all or part of the pore, thus leading to an energetic barrier to ion conduction. This process, termed “hydrophobic gating”, was first observed in molecular dynamics simulations of model nanopores, where the principles underlying hydrophobic gating (i.e., changes in diameter, polarity, or transmembrane voltage) have now been extensively validated. Computational, structural, and functional studies now indicate that biological ion channels may also exploit hydrophobic gating to regulate ion flow within their pores. Here we review the evidence for this process and propose that this unusual behavior of water represents an increasingly important element in understanding the relationship between ion channel structure and function.
    Graphical abstract




    Categories: Journal Articles
  • Mapping the Gating and Permeation Pathways in the Voltage-Gated Proton Channel Hv1
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Adam Chamberlin , Feng Qiu , Yibo Wang , Sergei Y. Noskov , H. Peter Larsson

    Voltage-gated proton channels (Hv1) are ubiquitous throughout nature and are implicated in numerous physiological processes. The gene encoding for Hv1, however, was only identified in 2006. The lack of sufficient structural information of this channel has hampered the understanding of the molecular mechanism of channel activation and proton permeation. This study uses both simulation and experimental approaches to further develop existing models of the Hv1 channel. Our study provides insights into features of channel gating and proton permeation pathway. We compare open- and closed-state structures developed previously with a recent crystal structure that traps the channel in a presumably closed state. Insights into gating pathways were provided using a combination of all-atom molecular dynamics simulations with a swarm of trajectories with the string method for extensive transition path sampling and evolution. A detailed residue–residue interaction profile and a hydration profile were studied to map the gating pathway in this channel. In particular, it allows us to identify potential intermediate states and compare them to the experimentally observed crystal structure of Takeshita et al. (Takeshita K, Sakata S, Yamashita E, Fujiwara Y, Kawanabe A, Kurokawa T, et al. X-ray crystal structure of voltage-gated proton channel. Nature 2014). The mechanisms governing ion transport in the wild-type and mutant Hv1 channels were studied by a combination of electrophysiological recordings and free energy simulations. With these results, we were able to further refine ideas about the location and function of the selectivity filter. The refined structural models will be essential for future investigations of this channel and the development of new drugs targeting cellular proton transport.
    Graphical abstract Highlights Our study reports on basic biophysical principles governing selective ion permeation in voltage-gated proton channels, which are membrane proteins with important roles in immune response and fertility. To further confirm and develop our model, we compared it to recently reported crystal structures. To gain further insight into the mechanisms of ion selectivity in these channels, we performed in vitro and in silico mutations on the channels and investigated their functioning. We found that targeted modifications around the constriction zone formed in an open state of the channel dramatically affect ion selectivity of the channel enabling transport of Na+. We also further investigate the gating behavior of the wild-type structures. Our in silico predictions were confirmed experimentally with regard to both the mutant and the wild-type structures, further establishing the validity of the channel model for future applications in drug development targeting this voltage-gated proton channel.




    Categories: Journal Articles
  • Combining Single-Molecule Imaging and Single-Channel Electrophysiology
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Eve E. Weatherill , Mark I. Wallace

    Combining simultaneous single-molecule fluorescence measurements of ion channel conformational change with single-channel electrophysiology would enable a direct link between structure and function. Such methods would help us to create a truly molecular “movie” of how these important biomolecules work. Here we review past and recent progress toward this goal.
    Graphical abstract




    Categories: Journal Articles
  • From Foe to Friend: Using Animal Toxins to Investigate Ion Channel Function
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Jeet Kalia , Mirela Milescu , Juan Salvatierra , Jordan Wagner , Julie K. Klint , Glenn F. King , Baldomero M. Olivera , Frank Bosmans

    Ion channels are vital contributors to cellular communication in a wide range of organisms, a distinct feature that renders this ubiquitous family of membrane-spanning proteins a prime target for toxins found in animal venom. For many years, the unique properties of these naturally occurring molecules have enabled researchers to probe the structural and functional features of ion channels and to define their physiological roles in normal and diseased tissues. To illustrate their considerable impact on the ion channel field, this review will highlight fundamental insights into toxin–channel interactions and recently developed toxin screening methods and practical applications of engineered toxins.
    Graphical abstract




    Categories: Journal Articles
  • Binding of ArgTX-636 in the NMDA Receptor Ion Channel
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Mette H. Poulsen , Jacob Andersen , Rune Christensen , Kasper B. Hansen , Stephen F. Traynelis , Kristian Strømgaard , Anders Skov Kristensen

    The N-methyl-d-aspartate receptors (NMDARs) constitute an important class of ligand-gated cation channels that are involved in the majority of excitatory neurotransmission in the human brain. Compounds that bind in the NMDAR ion channel and act as blockers are use- and voltage-dependent inhibitors of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker argiotoxin-636 (ArgTX-636) at recombinant GluN1/2A receptors to provide detailed information on the mechanism of block. The predicted binding site of ArgTX-636 is in the pore region of the NMDAR ion channel formed by residues in the transmembrane M3 and the M2 pore-loop segments of the GluN1 and GluN2A subunits. To assess the predicted binding mode in further detail, we performed an alanine- and glycine-scanning mutational analysis of this pore-loop segment to systematically probe the role of pore-lining M2 residues in GluN1 and GluN2A in the channel block by ArgTX-636. Comparison of M2 positions in GluN1 and GluN2A where mutation influences ArgTX-636 potency suggests differential contribution of the M2-loops of GluN1 and GluN2A to binding of ArgTX-636. The results of the mutational analysis are highly relevant for the future structure-based development of argiotoxin-derived NMDAR channel blockers.
    Graphical abstract




    Categories: Journal Articles
  • Ion Channel Engineering: Perspectives and Strategies
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Prakash Subramanyam , Henry M. Colecraft

    Ion channels facilitate the passive movement of ions down an electrochemical gradient and across lipid bilayers in cells. This phenomenon is essential for life and underlies many critical homeostatic processes in cells. Ion channels are diverse and differ with respect to how they open and close (gating) and to their ionic conductance/selectivity (permeation). Fundamental understanding of ion channel structure–function mechanisms, their physiological roles, how their dysfunction leads to disease, their utility as biosensors, and development of novel molecules to modulate their activity are important and active research frontiers. In this review, we focus on ion channel engineering approaches that have been applied to investigate these aspects of ion channel function, with a major emphasis on voltage-gated ion channels.
    Graphical abstract




    Categories: Journal Articles
  • Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties
    [Jan 2015]

    Publication date: 16 January 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 1

    Author(s): Martin K.M. Engqvist , R. Scott McIsaac , Peter Dollinger , Nicholas C. Flytzanis , Michael Abrams , Stanford Schor , Frances H. Arnold

    Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal-binding pocket of Gloeobacter violaceus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to ±80nm, extending the protein's light absorption significantly beyond the range of known natural PPRs. While proton-pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurred blue and red shifts of 42nm and 22nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty eight of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the WT (wild-type) protein.
    Graphical abstract




    Categories: Journal Articles