A New General Method for Simultaneous Fitting of Temperature- and Concentration-Dependence of Reaction Rates Yields Kinetic and Thermodynamic Parameters for HIV Reverse Transcriptase Specificity [DNA and Chromosomes]

March 2nd, 2017 by An Li, Jessica L. Ziehr, Kenneth A. Johnson

Recent studies have demonstrated the dominant role of induced-fit in enzyme specificity of HIV reverse transcriptase and many other enzymes. However, relevant thermodynamic parameters are lacking and equilibrium thermodynamic methods are of no avail because the key parameters can only determined by kinetic measurement. By modifying KinTek Explorer software, we present a new general method for globally fitting data collected over a range of substrate concentrations and temperatures and apply it to HIV reverse transcriptase. Fluorescence stopped-flow methods were used to record the kinetics of enzyme conformational changes that monitor nucleotide binding and incorporation. The nucleotide concentration dependence was measured at temperatures ranging from 5 to 37C and the raw data were fit globally to derive a single set of rate constants at 37C and a set of activation enthalpy terms to account for the kinetics at all other temperatures. This comprehensive analysis afforded thermodynamic parameters for nucleotide binding (Kd, ΔG, ΔH, ΔS at 37C), and kinetic parameters for enzyme conformational changes and chemistry (rate constants and activation enthalpy). Comparisons between wild-type enzyme and a mutant resistant to nucleoside analogs used to treat HIV infections reveal that the ground state binding is weaker and the activation enthalpy for the conformational change step is significantly larger for the mutant. Further studies to explore the structural underpinnings of the observed thermodynamics and kinetics of the conformational change step may help to design better analogs to treat HIV infections and other diseases. Our new method is generally applicable to enzyme and chemical kinetics.
  • Posted in Journal of Biological Chemistry, Publications
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Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11

February 28th, 2017 by Jing Li

Nature Chemical Biology 13, 486 (2017). doi:10.1038/nchembio.2326

Authors: Jing Li, Tanya Yakushi, Francesco Parlati, Andrew L Mackinnon, Christian Perez, Yuyong Ma, Kyle P Carter, Sharon Colayco, Gavin Magnuson, Brock Brown, Kevin Nguyen, Stefan Vasile, Eigo Suyama, Layton H Smith, Eduard Sergienko, Anthony B Pinkerton, Thomas D Y Chung, Amy E Palmer, Ian Pass, Sonja Hess, Seth M Cohen & Raymond J Deshaies

  • Posted in Nat Chem Biol, Publications
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A fully automated flow-based approach for accelerated peptide synthesis

February 28th, 2017 by Alexander J Mijalis

Nature Chemical Biology 13, 464 (2017). doi:10.1038/nchembio.2318

Authors: Alexander J Mijalis, Dale A Thomas, Mark D Simon, Andrea Adamo, Ryan Beaumont, Klavs F Jensen & Bradley L Pentelute

Here we report a fully automated, flow-based approach to solid-phase polypeptide synthesis, with amide bond formation in 7 seconds and total synthesis times of 40 seconds per amino acid residue. Crude peptide purities and isolated yields were comparable to those for standard-batch solid-phase peptide synthesis. At full capacity, this approach can yield tens of thousands of individual 30-mer peptides per year.

  • Posted in Nat Chem Biol, Publications
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A new genome-mining tool redefines the lasso peptide biosynthetic landscape

February 28th, 2017 by Jonathan I Tietz

Nature Chemical Biology 13, 470 (2017). doi:10.1038/nchembio.2319

Authors: Jonathan I Tietz, Christopher J Schwalen, Parth S Patel, Tucker Maxson, Patricia M Blair, Hua-Chia Tai, Uzma I Zakai & Douglas A Mitchell

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The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA

February 28th, 2017 by Rodolfo Zentella

Nature Chemical Biology 13, 479 (2017). doi:10.1038/nchembio.2320

Authors: Rodolfo Zentella, Ning Sui, Benjamin Barnhill, Wen-Ping Hsieh, Jianhong Hu, Jeffrey Shabanowitz, Michael Boyce, Neil E Olszewski, Pei Zhou, Donald F Hunt & Tai-ping Sun

  • Posted in Nat Chem Biol, Publications
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Synthetic Riboswitches: From Plug and Pray toward Plug and Play

February 24th, 2017 by Maja Etzel and Mario Mörl

TOC Graphic

Biochemistry
DOI: 10.1021/acs.biochem.6b01218

Expanding the product portfolio of fungal type I fatty acid synthases

February 20th, 2017 by Zhiwei Zhu

Nature Chemical Biology 13, 360 (2017). doi:10.1038/nchembio.2301

Authors: Zhiwei Zhu, Yongjin J Zhou, Anastasia Krivoruchko, Martin Grininger, Zongbao K Zhao & Jens Nielsen

Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes into the reaction chambers and demonstrated their capability to convert acyl-ACP or acyl-CoA from canonical fatty acid biosynthesis to short/medium-chain fatty acids and methyl ketones.

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Engineering fatty acid synthases for directed polyketide production

February 20th, 2017 by Jan Gajewski

Nature Chemical Biology 13, 363 (2017). doi:10.1038/nchembio.2314

Authors: Jan Gajewski, Floris Buelens, Sascha Serdjukow, Melanie Janßen, Niña Cortina, Helmut Grubmüller & Martin Grininger

In this study, we engineered fatty acid synthases (FAS) for the biosynthesis of short-chain fatty acids and polyketides, guided by a combined in vitro and in silico approach. Along with exploring the synthetic capability of FAS, we aim to build a foundation for efficient protein engineering, with the specific goal of harnessing evolutionarily related megadalton-scale polyketide synthases (PKS) for the tailored production of bioactive natural compounds.

Ligand-promoted protein folding by biased kinetic partitioning

February 20th, 2017 by Karan S Hingorani

Nature Chemical Biology 13, 369 (2017). doi:10.1038/nchembio.2303

Authors: Karan S Hingorani, Matthew C Metcalf, Derrick T Deming, Scott C Garman, Evan T Powers & Lila M Gierasch

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.

G-Protein-coupled receptors: Decoding mixed signals

February 15th, 2017 by Thomas J Gardella

Nature Chemical Biology 13, 247 (2017). doi:10.1038/nchembio.2316

Author: Thomas J Gardella

A new mechanism of functional crosstalk between two distinct G-protein-coupled receptors (GPCRs)—the parathyroid hormone receptor (PTHR) and β2-adrenergic receptor (β2 Ar)—that occurs at the level of G protein βγ subunits and a specific adenylyl cyclase isoform is identified. This crosstalk augments cAMP signaling by the PTHR from endosomes, and thus promotes the actions of PTH ligands in bone target cells.