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Author Dzierlenga, M.W.; Antoniou, D.; Schwartz, S.D. doi  openurl
  Title Another Look at the Mechanisms of Hydride Transfer Enzymes with Quantum and Classical Transition Path Sampling Type Journal Article
  Year 2015 Publication Abbreviated Journal The Journal of Physical Chemistry Letters  
  Volume 6 Issue 7 Pages 1177-1181  
  Keywords  
  Abstract The mechanisms involved in enzymatic hydride transfer have been studied for years, but questions remain due, in part, to the difficulty of probing the effects of protein motion and hydrogen tunneling. In this study, we use transition path sampling (TPS) with normal mode centroid molecular dynamics (CMD) to calculate the barrier to hydride transfer in yeast alcohol dehydrogenase (YADH) and human heart lactate dehydrogenase (LDH). Calculation of the work applied to the hydride allowed for observation of the change in barrier height upon inclusion of quantum dynamics. Similar calculations were performed using deuterium as the transferring particle in order to approximate kinetic isotope effects (KIEs). The change in barrier height in YADH is indicative of a zero-point energy (ZPE) contribution and is evidence that catalysis occurs via a protein compression that mediates a near-barrierless hydride transfer. Calculation of the KIE using the difference in barrier height between the hydride and deuteride agreed well with experimental results.  
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  ISSN 1948-7185 ISBN Medium  
  Area Expedition Conference  
  Notes NULL Times cited: 21 Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46386  
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Author Wan, C.; Borgeson, B.; Phanse, S.; Tu, F.; Drew, K.; Clark, G.; Xiong, X.; Kagan, O.; Kwan, J.; Bezginov, A.; Chessman, K.; Pal, S.; Cromar, G.; Papoulas, O.; Ni, Z.; Boutz, D.R.; Stoilova, S.; Havugimana, P.C.; Guo, X.; Malty, R.H.; Sarov, M.; Greenblatt, J.; Babu, M.; Derry, W.B.; R. Tillier, E.; Wallingford, J.B.; Parkinson, J.; Marcotte, E.M.; Emili, A. doi  openurl
  Title Panorama of ancient metazoan macromolecular complexes Type Journal Article
  Year 2015 Publication Abbreviated Journal Nature  
  Volume 525 Issue 7569 Pages 339-344  
  Keywords Animals Biochemical Datasets as Topic Evolution Fractionation Humans Metazoa Molecular Multiprotein Complexes Multiprotein Complexes: chemistry Multiprotein Complexes: metabolism Protein Interaction Mapping Protein Interaction Maps Protein complexes Proteomics Reproducibility of Results Systems Biology Tandem Mass Spectrometry  
  Abstract Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, here we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we generated a draft conservation map consisting of more than one million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering reveals a spectrum of conservation, ranging from ancient eukaryotic assemblies that have probably served cellular housekeeping roles for at least one billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, affinity purification and functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic importance and adaptive value to animal cell systems.  
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  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 0028-0836 ISBN Medium  
  Area Expedition Conference  
  Notes Times cited: 288 Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46421  
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Author Hein, M. Y.; Hubner, N. C.; Poser, I.; Cox, J.; Nagaraj, N.; Toyoda, Y.; Gak, I. A.; Weisswange, I.; Mansfeld, J.; Buchholz, F.; Hyman, A. A.; Mann, M. doi  openurl
  Title A Human Interactome in Three Quantitative Dimensions Organized by Stoichiometries and Abundances Type Journal Article
  Year 2015 Publication Abbreviated Journal Cell  
  Volume 163 Issue 3 Pages 712-723  
  Keywords  
  Abstract Summary The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins and introduces a framework for quantitative network analysis.  
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  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 1097-4172 (Electronic)\textbackslash\r0092-8674 (Linking) ISBN Medium  
  Area Expedition Conference  
  Notes Times cited: 690 Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46438  
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Author Masterson, J.E.; Schwartz, S.D. doi  openurl
  Title Evolution Alters the Enzymatic Reaction Coordinate of Dihydrofolate Reductase Type Journal Article
  Year 2015 Publication Abbreviated Journal The Journal of Physical Chemistry B  
  Volume 119 Issue 3 Pages 989-996  
  Keywords  
  Abstract How evolution has affected enzyme function is a topic of great interest in the field of biophysical chemistry. Evolutionary changes from Escherichia coli dihydrofolate reductase (ecDHFR) to human dihydrofolate reductase (hsDHFR) have resulted in increased catalytic efficiency and an altered dynamic landscape in the human enzyme. Here, we show that a subpicosecond protein motion is dynamically coupled to hydride transfer catalyzed by hsDHFR but not ecDHFR. This motion propagates through residues that correspond to mutational events along the evolutionary path from ecDHFR to hsDHFR. We observe an increase in the variability of the transition states, reactive conformations, and times of barrier crossing in the human system. In the hsDHFR active site, we detect structural changes that have enabled the coupling of fast protein dynamics to the reaction coordinate. These results indicate a shift in the DHFR family to a form of catalysis that incorporates rapid protein dynamics and a concomitant shift to a more flexible path through reactive phase space.  
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  Language Summary Language Original Title  
  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 1520-6106 ISBN Medium  
  Area Expedition Conference  
  Notes NULL Times cited: 12 Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46517  
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Author Pan, X.; Schwartz, S.D. url  openurl
  Title Free energy surface of the Michaelis complex of lactate dehydrogenase: a network analysis of microsecond simulations Type Journal Article
  Year 2015 Publication Abbreviated Journal J Phys Chem B  
  Volume 119 Issue 17 Pages 5430-5436  
  Keywords  
  Abstract It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD(+)). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.  
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  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 1520-6106 ISBN Medium  
  Area Expedition Conference  
  Notes Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46553  
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Author Yogurtcu, O.N.; Johnson, M.E. url  openurl
  Title Theory of bi-molecular association dynamics in 2D for accurate model and experimental parameterization of binding rates Type Journal Article
  Year 2015 Publication Abbreviated Journal J Chem Phys  
  Volume 143 Issue 8 Pages 084117  
  Keywords Jhu, Fpr, Cme  
  Abstract The dynamics of association between diffusing and reacting molecular species are routinely quantified using simple rate-equation kinetics that assume both well-mixed concentrations of species and a single rate constant for parameterizing the binding rate. In two-dimensions (2D), however, even when systems are well-mixed, the assumption of a single characteristic rate constant for describing association is not generally accurate, due to the properties of diffusional searching in dimensions d ≤ 2. Establishing rigorous bounds for discriminating between 2D reactive systems that will be accurately described by rate equations with a single rate constant, and those that will not, is critical for both modeling and experimentally parameterizing binding reactions restricted to surfaces such as cellular membranes. We show here that in regimes of intrinsic reaction rate (ka) and diffusion (D) parameters ka/D > 0.05, a single rate constant cannot be fit to the dynamics of concentrations of associating species independently of the initial conditions. Instead, a more sophisticated multi-parametric description than rate-equations is necessary to robustly characterize bimolecular reactions from experiment. Our quantitative bounds derive from our new analysis of 2D rate-behavior predicted from Smoluchowski theory. Using a recently developed single particle reaction-diffusion algorithm we extend here to 2D, we are able to test and validate the predictions of Smoluchowski theory and several other theories of reversible reaction dynamics in 2D for the first time. Finally, our results also mean that simulations of reactive systems in 2D using rate equations must be undertaken with caution when reactions have ka/D > 0.05, regardless of the simulation volume. We introduce here a simple formula for an adaptive concentration dependent rate constant for these chemical kinetics simulations which improves on existing formulas to better capture non-equilibrium reaction dynamics from dilute to dense systems.  
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  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 0021-9606 ISBN Medium  
  Area Expedition Conference  
  Notes Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46618  
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Author Świderek, K.; Tuñón, I.; Martí, S.; Moliner, V. url  openurl
  Title Protein Conformational Landscapes and Catalysis. Influence of Active Site Conformations in the Reaction Catalyzed by L-Lactate Dehydrogenase Type Journal Article
  Year 2015 Publication Abbreviated Journal ACS Catal  
  Volume 5 Issue 4 Pages 1172-1185  
  Keywords KIEs LDH QM/MM free energy surfaces reaction mechanism single-molecule experiments  
  Abstract In the last decade L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both clinical diagnosis and in monitoring the course of many human diseases. It has been assumed from the 80s that the full catalytic process of LDH starts with the binding of the cofactor and the substrate followed by the enclosure of the active site by a mobile loop of the protein before the reaction to take place. In this paper we show that the chemical step of the LDH catalyzed reaction can proceed within the open loop conformation, and the different reactivity of the different protein conformations would be in agreement with the broad range of rate constants measured in single molecule spectrometry studies. Starting from a recently solved X-ray diffraction structure that presented an open loop conformation in two of the four chains of the tetramer, QM/MM free energy surfaces have been obtained at different levels of theory. Depending on the level of theory used to describe the electronic structure, the free energy barrier for the transformation of pyruvate into lactate with the open conformation of the protein varies between 12.9 and 16.3 kcal/mol, after quantizing the vibrations and adding the contributions of recrossing and tunneling effects. These values are very close to the experimentally deduced one (14.2 kcal·mol^-1^) and ~2 kcal·mol^-1^ smaller than the ones obtained with the closed loop conformer. Calculation of primary KIEs and IR spectra in both protein conformations are also consistent with our hypothesis and in agreement with experimental data. Our calculations suggest that the closure of the active site is mainly required for the inverse process; the oxidation of lactate to pyruvate. According to this hypothesis H4 type LDH enzyme molecules, where it has been propose that lactate is transformed into pyruvate, should have a better ability to close the mobile loop than the M4 type LDH molecules.  
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  Series Volume Series Issue Edition  
  ISSN 2155-5435 ISBN Medium  
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  Notes Null Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46623  
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Author Park, B.-C.; Yim, Y.-I.; Zhao, X.; Olszewski, M.B.; Eisenberg, E.; Greene, L.E. doi  openurl
  Title The clathrin-binding and J-domains of GAK support the uncoating and chaperoning of clathrin by Hsc70 in the brain Type Journal Article
  Year 2015 Publication Abbreviated Journal Journal of Cell Science  
  Volume 128 Issue 20 Pages 3811-3821  
  Keywords JHU, CME, hsc70  
  Abstract Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones.  
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  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 1477-9137 0021-9533 ISBN Medium  
  Area Expedition Conference  
  Notes Times cited: 15 Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46698  
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Author Robinson, M.S. openurl 
  Title Forty Years of Clathrin-coated Vesicles Type Journal Article
  Year 2015 Publication Abbreviated Journal Traffic  
  Volume 16 Issue 12 Pages 1210-1238  
  Keywords JHU, CME, review  
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  Notes disease information Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46711  
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Author Zhuo, Y.; Cano, K.E.; Wang, L.; Ilangovan, U.; Hinck, A.P.; Sousa, R.; Lafer, E.M. url  openurl
  Title Nuclear Magnetic Resonance Structural Mapping Reveals Promiscuous Interactions between Clathrin-Box Motif Sequences and the N-Terminal Domain of the Clathrin Heavy Chain Type Journal Article
  Year 2015 Publication Abbreviated Journal Biochemistry  
  Volume 54 Issue 16 Pages 2571-2580  
  Keywords JHU, CME, clathrin box  
  Abstract The recruitment and organization of clathrin at endocytic sites first to form coated pits and then clathrin-coated vesicles depend on interactions between the clathrin N-terminal domain (TD) and multiple clathrin binding sequences on the cargo adaptor and accessory proteins that are concentrated at such sites. Up to four distinct protein binding sites have been proposed to be present on the clathrin TD, with each site proposed to interact with a distinct clathrin binding motif. However, an understanding of how such interactions contribute to clathrin coat assembly must take into account observations that any three of these four sites on clathrin TD can be mutationally ablated without causing loss of clathrin-mediated endocytosis. To take an unbiased approach to mapping binding sites for clathrin-box motifs on clathrin TD, we used isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy. Our ITC experiments revealed that a canonical clathrin-box motif peptide from the AP-2 adaptor binds to clathrin TD with a stoichiometry of 3:1. Assignment of 90% of the total visible amide resonances in the TROSY-HSQC spectrum of (13)C-, (2)H-, and (15)N-labeled TD40 allowed us to map these three binding sites by analyzing the chemical shift changes as clathrin-box motif peptides were titrated into clathrin TD. We found that three different clathrin-box motif peptides can each simultaneously bind not only to the previously characterized clathrin-box site but also to the W-box site and the β-arrestin splice loop site on a single TD. The promiscuity of these binding sites can help explain why their mutation does not lead to larger effects on clathrin function and suggests a mechanism by which clathrin may be transferred between different proteins during the course of an endocytic event.  
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  Series Editor Series Title Abbreviated Series Title  
  Series Volume Series Issue Edition  
  ISSN 1520-4995 0006-2960 ISBN Medium  
  Area Expedition Conference  
  Notes each CLTC.TD binds three AP2B2s w/ KD of 420 uM Approved no  
  Call Number (up) AG @ matthewjvarga @ Serial 46732  
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