Journal of Molecular Biology

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  • Dynamic and Thermodynamic Response of the Ras Protein Cdc42Hs upon Association with the Effector Domain of PAK3
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Veronica R. Moorman , Kathleen G. Valentine , Sabrina Bédard , Vignesh Kasinath , Jakob Dogan , Fiona M. Love , A. Joshua Wand

    Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs–PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately −10kcalmol−1 at 298K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding.
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    Categories: Journal Articles
  • DNA Looping Provides for “Intersegmental Hopping” by Proteins: A Mechanism for Long-Range Site Localization
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Adam J. Pollak , Aaron T. Chin , Frank L.H. Brown , Norbert O. Reich

    Studies on how transcription factors and DNA modifying enzymes passively locate specific sites on DNA have yet to be reconciled with a sufficient set of mechanisms that can adequately account for the efficiency and speed of this process. This is especially true when considering that these DNA binding/modifying proteins have diverse levels of both cellular copy numbers and genomic recognition site densities. The monomeric bacterial DNA adenine methyltransferase (Dam) is responsible for the rapid methylation of the entire chromosome (with only ~100 Dam copies per cell) and the regulated methylation of closely spaced sites that controls the expression of virulence genes in several human pathogens. Provocatively, we find that Dam travels between its recognition sites most efficiently when those sites are ~500bp apart. We propose that this is manifested by Dam moving between distal regions on the same DNA molecule, which is mediated by DNA looping, a phenomenon we designate as intersegmental hopping. Importantly, an intermediate found in other systems including two simultaneously bound, looped DNA strands is not involved here. Our results suggest that intersegmental hopping contributes to enzymatic processivity (multiple modifications), which invoke recent reports demonstrating that DNA looping can assist in site finding. Intersegmental hopping is possibly used by other sequence-specific DNA binding proteins, such as transcription factors and regulatory proteins, given certain biological context. While a general form of this mechanism is proposed by many research groups, our consideration of DNA looping in the context of processive catalysis provides new mechanistic insights and distinctions.
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    Categories: Journal Articles
  • DNA Recognition by a σ54 Transcriptional Activator from Aquifex aeolicus
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Natasha K. Vidangos , Johanna Heideker , Artem Lyubimov , Meindert Lamers , Yixin Huo , Jeffrey G. Pelton , Jimmy Ton , Jay Gralla , James Berger , David E. Wemmer

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix–turn–helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DBD of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4-binding sites were identified upstream (−145 and −85bp) from the start of the lpxC gene, which is responsible for the first committed step in lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the −145-binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homolog, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base-specific contacts contributing to affinity than for Fis.
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    Categories: Journal Articles
  • Phosphorylation- and Nucleotide-Binding-Induced Changes to the Stability and Hydrogen Exchange Patterns of JNK1β1 Provide Insight into Its Mechanisms of Activation
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Gavin R. Owen , Stoyan Stoychev , Ikechukwu Achilonu , Heini W. Dirr

    Many studies have characterized how changes to the stability and internal motions of a protein during activation can contribute to their catalytic function, even when structural changes cannot be observed. Here, unfolding studies and hydrogen–deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1β1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide binding on both inactive and active JNK1β1 using the ATP analogue, 5ʹ-adenylyl-imidodiphosphate (AMP-PNP). JNK1β1 phosphorylation alters HX in regions involved in catalysis and substrate binding, changes that can be ascribed to functional modifications in either structure and/or backbone flexibility. Increased HX in the hinge between the N- and C-terminal domains implied that it acquires enhanced flexibility upon phosphorylation that may be a prerequisite for interdomain closure. In combination with the finding that nucleotide binding destabilizes the kinase, the patterns of solvent protection by AMP-PNP were consistent with a novel mode of nucleotide binding to the C-terminal domain of a destabilized and open domain conformation of inactive JNK1β1. Solvent protection by AMP-PNP of both N- and C-terminal domains in active JNK1β1 revealed that the domains close around nucleotide upon phosphorylation, concomitantly stabilizing the kinase. This suggests that phosphorylation activates JNK1β1 in part by increasing hinge flexibility to facilitate interdomain closure and the creation of a functional active site. By uncovering the complex interplay that occurs between nucleotide binding and phosphorylation, we present new insight into the unique mechanisms by which JNK1β1 is regulated.
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  • Characterization of the Grp94/OS-9 Chaperone–Lectin Complex
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Paul M. Seidler , Stephen A. Shinsky , Feng Hong , Zihai Li , Michael S. Cosgrove , Daniel T. Gewirth

    Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum (ER) of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER-associated degradation quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins. To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full-length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal mannose recognition homology lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains “mammalian-specific insets” that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate and it additionally hints at a plausible biological role for the Grp94/OS-9 complex.
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    Categories: Journal Articles
  • A Fibrin-Specific Monoclonal Antibody from a Designed Phage Display Library Inhibits Clot Formation and Localizes to Tumors In Vivo
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Alessia Putelli , Jonathan D. Kiefer , Matthias Zadory , Mattia Matasci , Dario Neri

    Fibrin formation from fibrinogen is a rare process in the healthy organism but is a pathological feature of thrombotic events, cancer and a wide range of inflammatory conditions. We have designed and constructed an antibody phage display library (containing 13 billion clones) for the selective recognition of the N-terminal peptide of fibrin alpha chain. The key structural feature for selective fibrin binding was a K94E mutation in the VH domain. From this library, an antibody was isolated (termed AP2), which recognizes the five N-terminal amino acids of fibrin with high affinity (K d =44nM), but does not bind to fibrinogen. The AP2 antibody could be expressed in various formats (scFv, small immune protein and IgG) and inhibited fibrin clot formation in a concentration-dependent manner. Moreover, the AP2 antibody stained the fibrin-rich provisional stroma in solid tumors but did not exhibit any detectable staining toward normal tissues. Using a radioiodinated antibody preparation and quantitative biodistribution studies in tumor-bearing mice, AP2 was shown to selectively localize to fibrin-rich F9 murine teratocarcinomas, but not to SKRC-52 human kidney cancer xenografts. Collectively, the experiments indicate that the AP2 antibody recognizes fibrin in vitro and in vivo. The antibody may facilitate the development of fibrin-specific therapeutic agents.
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    Categories: Journal Articles
  • Coevolution of Specificity Determinants in Eukaryotic Glutamyl- and Glutaminyl-tRNA Synthetases
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Andrew Hadd , John J. Perona

    The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNAGln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNAGln and tRNAGlu with glutamate. This ancient GluRS also separately differentiated to exclude tRNAGln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNAGln and tRNAGlu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.
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  • Crystal Structure of a Symmetric Football-Shaped GroEL:GroES2-ATP14 Complex Determined at 3.8Å Reveals Rearrangement between Two GroEL Rings
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Ayumi Koike-Takeshita , Takatoshi Arakawa , Hideki Taguchi , Tatsuro Shimamura

    The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES2 complex (the “football”-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES2-ATP14 complex from Escherichia coli at 3.8Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the “bullet” complex). However, the two GroES-bound GroEL rings form a modified interface by an ~7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.
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    Categories: Journal Articles
  • Functional Suppression of HAMP Domain Signaling Defects in the E. coli Serine Chemoreceptor
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Run-Zhi Lai , John S. Parkinson

    HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric Escherichia coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: unmodified E residues promote kinase-off output, and methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer 3 of the HAMP bundle and followed in vivo CheA activity with an assay based on Förster resonance energy transfer. We found that an alanine or a serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E and S255A) shifted Tsr output toward the kinase-off state, but two others (S255G and L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations.
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    Categories: Journal Articles
  • A Conserved Isoleucine Maintains the Inactive State of Bruton's Tyrosine Kinase
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Scott E. Boyken , Nikita Chopra , Qian Xie , Raji E. Joseph , Thomas E. Wales , D. Bruce Fulton , John R. Engen , Robert L. Jernigan , Amy H. Andreotti

    Despite high level of homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamics simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest that this isoleucine side chain in the Tec family kinases acts as a “wedge” that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain.
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    Categories: Journal Articles
  • Characterization of Membrane Protein Interactions by Isothermal Titration Calorimetry
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Alan J. Situ , Thomas Schmidt , Parichita Mazumder , Tobias S. Ulmer

    Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix–helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of −4.61±0.04kcal/mol at bicelle conditions where the sampling of random helix–helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of >1kcal/mol was obtained from helix–helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500±100s−1 and 2.1±0.1s−1, respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.
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    Categories: Journal Articles
  • Erratum for “Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase” [J. Mol. Biol. 426 (2014) 3028–3040]
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Inchul Shin , Kitae Han , Sangkee Rhee







    Categories: Journal Articles
  • An Ancient Autoproteolytic Domain Found in GAIN, ZU5 and Nucleoporin98
    [Nov 2014]

    Publication date: Available online 20 October 2014
    Source:Journal of Molecular Biology

    Author(s): Yuxing Liao , Jimin Pei , Hua Cheng , Nick V. Grishin

    A large family of G protein-coupled receptors (GPCRs) involved in cell adhesion has a characteristic autoproteolysis motif of HLT/S known as the GPCR proteolysis site (GPS). GPS is also shared by polycystic kidney disease proteins and it precedes the first transmembrane segment in both families. Recent structural studies have elucidated the GPS to be part of a larger domain named GPCR autoproteolysis inducing (GAIN) domain. Here we demonstrate the remote homology relationships of GAIN domain to ZU5 domain and Nucleoporin98 (Nup98) C-terminal domain by structural and sequence analysis. Sequence homology searches were performed to extend ZU5-like domains to bacteria and archaea, as well as new eukaryotic families. We found that the consecutive ZU5-UPA-death domain domain organization is commonly used in human cytoplasmic proteins with ZU5 domains, including CARD8 (caspase recruitment domain-containing protein 8) and NLRP1 (NACHT, LRR and PYD domain-containing protein 1) from the FIIND (Function to Find) family. Another divergent family of extracellular ZU5-like domains was identified in cartilage intermediate layer proteins and FAM171 proteins. Current diverse families of GAIN domain subdomain B, ZU5 and Nup98 C-terminal domain likely evolved from an ancient autoproteolytic domain with an HFS motif. The autoproteolytic site was kept intact in Nup98, p53-induced protein with a death domain and UNC5C-like, deteriorated in many ZU5 domains and changed in GAIN and FIIND. Deletion of the strand after the cleavage site was observed in zonula occluden-1 and some Nup98 homologs. These findings link several autoproteolytic domains, extend our understanding of GAIN domain origination in adhesion GPCRs and provide insights into the evolution of an ancient autoproteolytic domain.
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    Categories: Journal Articles
  • TMEM16 proteins: Unknown structure and confusing functions
    [Nov 2014]

    Publication date: Available online 17 October 2014
    Source:Journal of Molecular Biology

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

    The TMEM16 family of membrane proteins, also known as anoctamins, play 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 (CaCCs) and are important for transepithelial ion transport, olfaction, phototransduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. The role(s) 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 transport. 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.
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    Categories: Journal Articles
  • Lysophospholipid-Containing Membranes Modulate the Fibril Formation of the Repeat Domain of a Human Functional Amyloid, Pmel17
    [Nov 2014]

    Publication date: Available online 14 October 2014
    Source:Journal of Molecular Biology

    Author(s): Zhiping Jiang , Jennifer C. Lee

    Pmel17 is an important protein for pigmentation in human skin and eyes. Proteolytic fragments from Pmel17 form fibrils upon which melanin is deposited in melanosomes. The repeat domain (RPT) derived from Pmel17 only forms fibrils under acidic melanosomal conditions. Here, we examined the effects of lipids on RPT aggregation to explore whether intramelanosomal vesicles can facilitate fibrillogenesis. Using transmission electron microscopy, circular dichroism, and fluorescence spectroscopy, we monitored fibril formation at the ultrastructural, secondary conformational, and local levels, respectively. Phospholipid vesicles and lysophospholipid (lysolipid) micelles were employed as membrane mimics. The surfactant-like lysolipids are particularly pertinent due to their high content in melanosomal membranes. Interestingly, RPT aggregation kinetics were influenced only by lysolipid-containing phospholipid vesicles. While both vesicles containing either anionic lysophosphatidylglycerol (LPG) or zwitterionic lysophosphatidylcholine (LPC) stimulate aggregation, LPG exerted a greater effect on reducing the apparent nucleation time. A detailed comparison showed distinct behaviors of LPG versus LPC monomers and micelles plausibly originating from their headgroup hydrogen bonding capabilities. Acceleration and retardation of aggregation were observed for LPG monomers and micelles, respectively. Because a specific interaction between LPG and RPT was identified by intrinsic W423 fluorescence and induced α-helical structure, it is inferred that binding of LPG near the C-terminal amyloid core initiates intermolecular association, whereas stabilization of α-helical conformation inhibits β-sheet formation. Contrastingly, LPC promotes RPT aggregation at both submicellar and micellar concentrations via non-specific binding with undetectable secondary structural change. Our findings suggest that protein–lysolipid interactions within melanosomes may regulate amyloid formation in vivo.
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    Categories: Journal Articles
  • The CamSol Method of Rational Design of Protein Mutants with Enhanced Solubility
    [Nov 2014]

    Publication date: Available online 14 October 2014
    Source:Journal of Molecular Biology

    Author(s): Pietro Sormanni , Francesco A. Aprile , Michele Vendruscolo

    Protein solubility is often an essential requirement in biotechnological and biomedical applications. Great advances have been made during the past decade in understanding the principles that determine this specific property of proteins, in particular those concerning the physicochemical characteristics of their constituent amino acids. By exploiting these advances, we present the CamSol method for the rational design of protein variants with enhanced solubility. The method works by performing a rapid computational screening of tens of thousand of mutations to identify those with the greatest impact on the solubility of the target protein while maintaining its native state and biological activity. The application to a single-domain antibody that targets the Alzheimer's A<beta>peptide demonstrates that the method predicts with great accuracy solubility changes upon mutation, thus offering a cost-effective strategy to help the production of soluble proteins for academic and industrial purposes.
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    Categories: Journal Articles
  • A ‘fuzzy’-logic language for encoding multiple physical traits in biomolecules
    [Nov 2014]

    Publication date: Available online 13 October 2014
    Source:Journal of Molecular Biology

    Author(s): Shira Warszawski , Ravit Netzer , Dan S. Tawfik , Sarel J. Fleishman

    To carry out their activities biological macromolecules balance different physical traits, such as stability, interaction affinity, and selectivity. How such often-opposing traits are encoded in a macromolecular system is critical to our understanding of evolutionary processes and ability to design new molecules with desired functions. We present a framework for constraining design simulations to balance different physical characteristics. Each trait is represented by the equilibrium fractional occupancy of the desired state relative to its alternatives, ranging from none to full occupancy, and the different traits are combined using Boolean operators to effect a ‘fuzzy’-logic language for encoding any combination of traits. In another paper, we presented a new combinatorial-backbone design algorithm AbDesign where the fuzzy-logic framework was used to optimize protein backbones and sequences for both stability and binding affinity in antibody-design simulation. We now extend this framework and find that fuzzy-logic design simulations reproduce sequence and structure design principles seen in nature to underlie exquisite specificity on the one hand, and multispecificity on the other. The fuzzy-logic language is broadly applicable and could help define the space of tolerated and beneficial mutations in natural biomolecular systems and design artificial molecules that encode complex characteristics.





    Categories: Journal Articles
  • Regional Discrimination and Propagation of Local Rearrangements Along the Ribosomal Exit Tunnel
    [Nov 2014]

    Publication date: Available online 13 October 2014
    Source:Journal of Molecular Biology

    Author(s): Jianli Lu , Carol Deutsch

    All proteins, from bacteria to man, are made in the ribosome and are elongated, one residue at a time, at the peptidyl transferase center (PTC). This growing peptide chain wends its way through the ribosomal tunnel to the exit port, ~ 100 angstroms from the PTC. We have identified locations in the tunnel that sense and respond to single side chains of the nascent peptide to induce local conformational changes. Moreover, side-chain sterics and rearrangements deep in the tunnel influence the disposition of residues 45Å away at the exit port and are consistent with side-chain induced axial retraction of the peptide backbone. These coupled responses are neither haphazard nor uniform along the tunnel. Rather, they are confined to discriminating zones in the tunnel and are sequence-specific. Such discerning communication may contribute to folding events and mechanisms governing sequence-specific signaling between different regions of the tunnel during translation.
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    Categories: Journal Articles
  • Non-native structure appears in microseconds during the folding of E. coli RNase H
    [Nov 2014]

    Publication date: Available online 13 October 2014
    Source:Journal of Molecular Biology

    Author(s): Laura E. Rosen , Sagar V. Kathuria , C. Robert Matthews , Osman Bilsel , Susan Marqusee

    The folding pathway of E. coli RNase H is one of the best experimentally characterized for any protein. In spite of this, spectroscopic studies have never captured the earliest events. Using continuous flow microfluidic mixing, we have now observed the first several milliseconds of folding by monitoring the tryptophan fluorescence lifetime (60 microsecond dead time). Two folding intermediates are observed, the second of which is the previously characterized Icore millisecond intermediate. The new, earlier intermediate is likely on-pathway and appears to have long-range non-native structure, providing a rare example of such non-native structure formation in a folding pathway. The tryptophan fluorescence lifetimes also suggest a deviation from native packing in the second intermediate, Icore. Similar results from a fragment of RNase H demonstrate that only half of the protein is significantly involved in this early structure formation. These studies give us a view of the formation of tertiary structure on the folding pathway, and complement previous hydrogen exchange studies which monitored only secondary structure and observed sequential native structure formation. Our results provide detailed folding information on both a timescale and a size-scale accessible to all-atom molecular dynamic simulations of protein folding.
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    Categories: Journal Articles
  • Molecular Interactions and Residues Involved in Force Generation in the T4 Viral DNA Packaging Motor
    [Nov 2014]

    Publication date: Available online 13 October 2014
    Source:Journal of Molecular Biology

    Author(s): Amy D. Migliori , Douglas E. Smith , Gaurav Arya

    Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations.
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    Categories: Journal Articles