Methods to Prepare DNA for Efficient Massive Sequencing
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Control of Enzyme Activity Using Chemical Rescue of Protein Structure
Control of Enzyme Activity using Chemical Rescue of Protein Structure by Yang Du A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science in Engineering Baltimore, Maryland May 2016 ii Abstract A cell can modulate the level of protein activity by the regulation of the amount of protein. On the other hand, strategies for controlling of cellular protein activity may lie in regulating the protein’s specific activity directly. Based on this idea, we demonstrated a method for designing an effector site directly into the catalytic domain of an enzyme and using a chemical compound to regulate the protein’s activity. This approach is different from the traditional chemical rescue of enzymes because it relies on disruption and restoration of protein structure instead of disrupting the active site and having the chemical compound restore the chemical functionality to the active site as a means to achieve modulated function. In detail, we demonstrate the activity of TEM-1 can be disrupted by removing buried tryptophan or leucine side chain that serve as a buttress supporting the protein structure. Three cases of site-direct mutations (W227G & W286G, L247G & W286G, and L282G & W286G) to TEM-1 β-lactamase enzyme (BLA) were made. In all cases, we observe disruption of enzyme function. To test the theory of chemical rescue of protein structure, we then assay β-lactamase activity in the presence of specific chemical compounds designed to bind in place of the iii side chains of the mutated amino acids. We observed an 8- fold restoration of W227G/W286G enzyme function in the presence of 2- (1-naphthylmethyl)-isoquinoline (NMIQ) and 3 -methyl-2- (1-naphthylmethyl)-1,3-benzothiazol-3- ium (MNMBT). -
DNA: the Timeline and Evidence of Discovery
1/19/2017 DNA: The Timeline and Evidence of Discovery Interactive Click and Learn (Ann Brokaw Rocky River High School) Introduction For almost a century, many scientists paved the way to the ultimate discovery of DNA and its double helix structure. Without the work of these pioneering scientists, Watson and Crick may never have made their ground-breaking double helix model, published in 1953. The knowledge of how genetic material is stored and copied in this molecule gave rise to a new way of looking at and manipulating biological processes, called molecular biology. The breakthrough changed the face of biology and our lives forever. Watch The Double Helix short film (approximately 15 minutes) – hyperlinked here. 1 1/19/2017 1865 The Garden Pea 1865 The Garden Pea In 1865, Gregor Mendel established the foundation of genetics by unraveling the basic principles of heredity, though his work would not be recognized as “revolutionary” until after his death. By studying the common garden pea plant, Mendel demonstrated the inheritance of “discrete units” and introduced the idea that the inheritance of these units from generation to generation follows particular patterns. These patterns are now referred to as the “Laws of Mendelian Inheritance.” 2 1/19/2017 1869 The Isolation of “Nuclein” 1869 Isolated Nuclein Friedrich Miescher, a Swiss researcher, noticed an unknown precipitate in his work with white blood cells. Upon isolating the material, he noted that it resisted protein-digesting enzymes. Why is it important that the material was not digested by the enzymes? Further work led him to the discovery that the substance contained carbon, hydrogen, nitrogen and large amounts of phosphorus with no sulfur. -
What Are the Preferred Ways for Proteins to Interact?
Principles of Protein−Protein Interactions: What are the Preferred Ways For Proteins To Interact? Ozlem Keskin,*,† Attila Gursoy,† Buyong Ma,‡ and Ruth Nussinov*,‡,§ Koc University, Center for Computational Biology and Bioinformatics and College of Engineering, Rumelifeneri Yolu, 34450 Sariyer Istanbul, Turkey; Basic Research Program, SAIC−Frederick, Inc., Center for Cancer Research Nanobiology Program, NCIsFrederick, Frederick, Maryland 21702; and Sackler Institute of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel Received July 13, 2007 Contents 7.3. Chemistry of the Interactions: How Are 15 Subtle Differences Distinguished? 1. Introduction 1 8. Allostery 15 − 1.1. Protein Protein Interactions: Toward 1 9. Large Assemblies 16 Functional Prediction and Drug Design 10. Crystal Interfaces 16 1.2. Proteins are Flexible Molecules Even Though 3 We Frequently Treat Them as Rigid 11. Concluding Remarks: Preferred Organization in 17 Protein Interactions 1.3. Proteins Interact through Their Surfaces 5 12. Acknowledgment 18 2. Cooperativity in Protein Folding and in 5 Protein−Protein Associations 13. References 18 3. Protein−Protein Interfaces Have Preferred 6 Organization 1. Introduction 3.1. Description of Protein−Protein Interfaces 6 3.2. Some Amino Acids at the Interface Are Hot 7 1.1. Protein−Protein Interactions: Toward Spots Since They Contribute Significantly to Functional Prediction and Drug Design the Stability of the Protein−Protein Association Proteins are the working horse of the cellular machinery. 3.3. Protein Binding Sites Can Be Described as 8 They are responsible for diverse functions ranging from Consisting of a Combination of molecular motors to signaling. -
Photograph 51, by Rosalind Franklin (1952) [1]
Published on The Embryo Project Encyclopedia (https://embryo.asu.edu) Photograph 51, by Rosalind Franklin (1952) [1] By: Hernandez, Victoria Keywords: X-ray crystallography [2] DNA [3] DNA Helix [4] On 6 May 1952, at King´s College London in London, England, Rosalind Franklin photographed her fifty-first X-ray diffraction pattern of deoxyribosenucleic acid, or DNA. Photograph 51, or Photo 51, revealed information about DNA´s three-dimensional structure by displaying the way a beam of X-rays scattered off a pure fiber of DNA. Franklin took Photo 51 after scientists confirmed that DNA contained genes [5]. Maurice Wilkins, Franklin´s colleague showed James Watson [6] and Francis Crick [7] Photo 51 without Franklin´s knowledge. Watson and Crick used that image to develop their structural model of DNA. In 1962, after Franklin´s death, Watson, Crick, and Wilkins shared the Nobel Prize in Physiology or Medicine [8] for their findings about DNA. Franklin´s Photo 51 helped scientists learn more about the three-dimensional structure of DNA and enabled scientists to understand DNA´s role in heredity. X-ray crystallography, the technique Franklin used to produce Photo 51 of DNA, is a method scientists use to determine the three-dimensional structure of a crystal. Crystals are solids with regular, repeating units of atoms. Some biological macromolecules, such as DNA, can form fibers suitable for analysis using X-ray crystallography because their solid forms consist of atoms arranged in a regular pattern. Photo 51 used DNA fibers, DNA crystals first produced in the 1970s. To perform an X-ray crystallography, scientists mount a purified fiber or crystal in an X-ray tube. -
Enzyme Substrate Binding Two Models Have Been Proposed to Explain How an Enzyme Binds Its Substrate: the Lock-And –Key Model and the Induced-Fit Model
ENZYMOLOGY 1 Introduction إعداد محمود بركات Enzymology 1 - Definition: Biologic (organic catalysts) polymers that catalyze the chemical reactions. - Nature: Enzymes are proteins except catalytic RNA molecules (Ribozymes) (Ribozymes: short segment of RNA molecule which act as enzyme in processing RNA molecules (From immature to mature RNA)) - Characteristics: i. Enzymes are neither consumed nor permanently altered as a consequence of their participation in a reaction. Throughout their life span ii. Highly efficient. iii. Extremely selective catalysts. Specific iv. Thermolabile. (Enzymes are affected by the change of temperature when the temperature increases the enzymes will denatured) v. Site specific. (Enzymes found in cytoplasm differ from those found in organelles or membrane) vi. High turnover number compared to inorganic catalysts and other organic catalysts (Turnover number: the number of substrate molecules that can be converted by one molecule of catalyst into product molecule in a unit time) - Inorganic Catalysts such as Ni, Pb, Pt. • Thermostable • Lower turnover number Nomenclature of enzymes (In most cases, enzyme names end in –ase) 1. The common name for a hydrolase is derived from the substrate. ➢ Urea: remove -a, replace with -ase = urease ➢ Lactose: remove - ose, replace with - ase = lactase 2. Other enzymes are named for the substrate and the reaction catalyzed. ➢ Lactate dehydrogenase ➢ Pyruvate decarboxylase 3. Some names are historical - no direct relationship to substrate or reaction type. (Digestive Enzymes) Catalase, Pepsin, Chymotrypsin, Trypsin Classification of Enzymes - Enzyme Commission (EC) – according to International Union of Biochemistry & Molecular Biology (IUBMB) - Each enzyme was given 4-digit numbes [1.2.3.4] 1st one of the 6 major classes of enzyme activity 2nd the subclass (type of substrate or bond cleaved) 3rd the sub-subclass (group acted upon, cofactor required, etc...) 4th a serial number… (order in which enzyme was added to list) The 6 Magor Classes are: 1) Oxidoreductases (EC.1) catalyze redox reactions. -
A Brief History of Genetics
A Brief History of Genetics A Brief History of Genetics By Chris Rider A Brief History of Genetics By Chris Rider This book first published 2020 Cambridge Scholars Publishing Lady Stephenson Library, Newcastle upon Tyne, NE6 2PA, UK British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Copyright © 2020 by Chris Rider All rights for this book reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner. ISBN (10): 1-5275-5885-1 ISBN (13): 978-1-5275-5885-4 Cover A cartoon of the double-stranded helix structure of DNA overlies the sequence of the gene encoding the A protein chain of human haemoglobin. Top left is a portrait of Gregor Mendel, the founding father of genetics, and bottom right is a portrait of Thomas Hunt Morgan, the first winner of a Nobel Prize for genetics. To my wife for her many years of love, support, patience and sound advice TABLE OF CONTENTS List of Figures.......................................................................................... viii List of Text Boxes and Tables .................................................................... x Foreword .................................................................................................. xi Acknowledgements ................................................................................. xiii Chapter 1 ................................................................................................... -
STS.003 the Rise of Modern Science Spring 2008
MIT OpenCourseWare http://ocw.mit.edu STS.003 The Rise of Modern Science Spring 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms. STS.003 Spring 2007 Keywords for Week 12 Lecture 20: Eugenics Darwin, Descent of Man (1871) Francis Galton, “eugenics” (1883) “the science of improving the stock” Jean Baptiste Lamarck Inheritance of Acquired Characteristics Richard Dugdale, The Jukes: A Study in Crime, Pauperism, Disease and Heredity (1874) Karl Pearson Galton Laboratory for National Eugenics Charles Davenport Cold Spring Harbor Eugenics Record Office Arthur Estabrook, The Jukes in 1915 (1916) Henry Goddard, The Kallikak Family: A Study in the Heredity of Feeble-mindedness (1912) Deborah Kallikak Race Suicide Fitter Family Contests Sterilization Laws Buck v. Bell, 1927 Carrie Buck Justice Oliver Wendell Holmes, Jr. “Three generations of imbeciles is enough” Quotes But are not our physical faculties and the strength, dexterity and acuteness of our senses, to be numbered among the qualities whose perfection in the individual may be transmitted? Observation of the various breeds of domestic animals inclines us to believe that they are, and we can confirm this by direct observation of the human race. Condorcet, Future Progress of the Human Spirit (1795) If a twentieth part of the cost and pains were spent in measures for the improvement of the human race that is spent on the improvement of the breed of horses and cattle, what a galaxy of genius we might create. Francis Galton, “Hereditary Character and Talent” (1864) Both sexes ought to refrain from marriage if they are in any marked degree inferior in body or mind but such hopes are Utopian and will never be even partially realized until the laws of inheritance are thoroughly known. -
Video Worksheet - Secret of Photo 51
Name __________________________________________ Period ________ Date _______________ Video Worksheet - Secret of Photo 51 We are watching the NOVA video entitled “Secret of Photo 51.” This video demonstrates the race to determine the structure of DNA during the 1940s and 1950s. Of particular interest through the video, we see how a female scientist, Rosalind Franklin, was of essential to the discovery. 1. In 1962, who was awarded the Nobel Prize for the discovery of the structure of DNA? (Please give three names) 2. Which of those three wrote the book “The Double Helix”? 3. How did he characterize Rosalind Franklin in his book? 4. What was Rosalind Franklin like as a child? 5. Where did she study physics and chemistry? 6. What city did Rosalind Franklin perfect her work in crystallography in? 7. Where in England is Rosalind Franklin offered a position? 8. What misunderstanding occurred between Franklin and Maurice Wilkins? 9. What was the environment like for a female scientist? What nicknames was Franklin given? 10. What two forms of DNA did Franklin discover? Name __________________________________________ Period ________ Date _______________ 11. Who is in the audience listening to Franklin’s talk? What does he want to do with her information? 12. Who gives away Franklin’s unpublished work? To whom does he give it to? 13. When did Franklin get her best picture? What did she title it? 14. The race is now in earnest. How do the “discoverers” come to their conclusion about the structure of DNA? What information did they need? 15. Did Franklin approve of the model in 1953? 16. -
Deciphering Molecular Mechanisms of Adverse Reactions of Drugs
UNIVERSITY OF CALIFORNIA, SAN DIEGO Deciphering Molecular Mechanisms of Adverse Reactions of Drugs A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioinformatics and Systems Biology by Yu-Chen Chen Committee in charge: Professor Ruben Abagyan, Chair Professor Grace M. Kuo, Co-Chair Professor Nuno F. Bandeira Professor Sanjoy Dasgupta Professor Lucila Ohno-Machado 2014 Copyright Yu-Chen Chen, 2014 All rights reserved. Signature Page The Dissertation of Yu-Chen Chen is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Co-Chair _____________________________________________________________________ Chair University of California, San Diego 2014 iii TABLE OF CONTENTS Table of Contents Signature Page ............................................................................................................... iii Table of Contents .......................................................................................................... iv List of Figures ................................................................................................................ ix List of Tables ............................................................................................................... -
Encyclopedia of Kimilsungia
1 Preface Love of flower is a noble trait peculiar to man. Flower brings fragrance, emotion and beauty to people. That is why they love it, and hope to live beautifully and pure-heartedly like it. At the same time, they express their wish and desire, happiness and hope by means of it, and want to bring their life into full bloom, picturing themselves in it. Kimilsungia, which was named by Sukarno, the first President of the Republic of Indonesia, reflecting the desire of the progressive people of the world, is loved by mankind not only because it is beautiful but also it is symbolic of the greatness of President Kim Il Sung. The editorial board issues Encyclopedia of Kimilsungia in reflection of the unanimous will of the Korean people and the world’s progressive people who are desirous to bloom Kimilsungia more beautifully and propagate it more widely on the occasion of the centenary of the birth of President Kim Il Sung. The book introduces in detail how Kimilsungia came into being in the world, its propagation, Kimilsungia festivals and exhibitions held in Korea and foreign countries every year, events held on the occasion of the anniversary of the naming of the flower, and its biological features and cultivating techniques the Korean botanists and growers have studied and perfected. And edited in the book are the typical literary works depicting Kimilsungia and some of gift plants presented to President Kim Il Sung by foreign countries. In addition, common knowledge of flower is compiled. The editorial board hopes this book will be a help to the flower lovers and people of other countries of the world who are eager to know and grow Kimilsungia. -
Structural Biochemistry/Enzyme/Active Site 1 Structural Biochemistry/Enzyme/Active Site
Structural Biochemistry/Enzyme/Active Site 1 Structural Biochemistry/Enzyme/Active Site Overview An active site is the part of an enzyme that directly binds to a substrate and carries a reaction. It contains catalytic groups which are amino acids that promote formation and degradation of bonds. By forming and breaking these bonds, enzyme and substrate interaction promotes the formation of the transition state structure. Enzymes help a reaction by stabilizing the transition state intermediate. This is accomplished by lowering the energy barrier or activation energy- the energy that is required to promote the formation of transition state intermediate. The three dimensional cleft is formed by the groups that come from different part of the amino acid sequences. The active site is only a small part of the total enzyme volume. It enhances the enzyme to bind to substrate and catalysis by many differnet weak interactions because of its nonpolar microenvironment. The weak interactions includes the Van der Waals, hydrogen bonding, and electrostatic interactions. The arrangement of atoms in the active site is crucial for binding spectificity. The overall result is the acceleration of the reaction process and increasing the rate of reaction. Furthermore, not only do enzymes contain catalytic abilities, but the active site also carries the recognition of substrate. The enzyme active site is the binding site for catalytic and inhibition reactions of enzyme and substrate; structure of active site and its chemical characteristic are of specific for the binding of a particular substrate. The binding of the substrate to the enzyme causes changes in the chemical bonds of the substrate and causes the reactions that lead to the formation of products. -
On the Specificity of Protein-Protein Interactions in the Context of Disorder
On the specificity of protein-protein interactions in the context of disorder Teilum, Kaare; Olsen, Johan G.; Kragelund, Birthe B. Published in: Biochemical Journal DOI: 10.1042/BCJ20200828 Publication date: 2021 Document version Publisher's PDF, also known as Version of record Document license: CC BY-NC-ND Citation for published version (APA): Teilum, K., Olsen, J. G., & Kragelund, B. B. (2021). On the specificity of protein-protein interactions in the context of disorder. Biochemical Journal, 478(11), 2035-2050. https://doi.org/10.1042/BCJ20200828 Download date: 29. sep.. 2021 Biochemical Journal (2021) 478 2035–2050 https://doi.org/10.1042/BCJ20200828 Review Article On the specificity of protein–protein interactions in the context of disorder Kaare Teilum1,2, Johan G. Olsen1,2,3 and Birthe B. Kragelund1,2,3 1The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark; 2Structural Biology and NMR Downloaded from http://portlandpress.com/biochemj/article-pdf/478/11/2035/914573/bcj-2020-0828c.pdf by Copenhagen University user on 24 June 2021 Laboratory, Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark; 3REPIN, Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark Correspondence: Birthe B. Kragelund ([email protected]) With the increased focus on intrinsically disordered proteins (IDPs) and their large interac- tomes, the question about their specificity — or more so on their multispecificity — arise. Here we recapitulate how specificity and multispecificity are quantified and address through examples if IDPs in this respect differ from globular proteins. The conclusion is that quantitatively, globular proteins and IDPs are similar when it comes to specificity.