Biochemical Studies to Probe the Domain-Domain Communication Pathways in E. coli Prolyl-tRNA Synthetase Heidi Schmit and Sanchita Hati Department of Chemistry,

Slides:



Advertisements
Similar presentations
Molecular Genetics DNA RNA Protein Phenotype Genome Gene
Advertisements

Basics of Molecular Biology
The Central Dogma (Francis Crick, 1958) (Transcription) (Translation) DNA  RNA  Protein (Gene/Genotype) (Phenotype) An informational process between.
translation RBS RBS: ribosome binding site Ribosome(r RNA + r protein)
Nuclease cleavage rRNA Operon 7 copies/genome in E. coli.
Chapter 22 (Part 1) Protein Synthesis. Translating the Message How does the sequence of mRNA translate into the sequence of a protein? What is the genetic.
2.7 DNA Replication, transcription and translation
Ribosome Structure 1. Outline the structure of a ribosome based on the diagram: ● A site.
Role of Protein Electrostatics on the Post-transfer Editing Function of Escherichia coli Prolyl-tRNA Synthetase Bach Cao †, Karl J. Meitzner †, Mathew.
Birth of proteins by translation
Components needed for Translation tRNAs Aminoacyl-tRNA synthetases Ribosomes.
Manufacture of Human Interleukin 13 Protein Using a Prokaryotic Expression System Ryan Rupp, York College of Pennsylvania, Department of Biological Sciences.
Role of Coupled-Domain Motions on the Catalytic Activity of Escherichia coli Prolyl- tRNA Synthetase Kurt Zimmerman †, Bach Cao †, Alexander Greene †,
Fig The Elongation Cycle (in prokaryotes).
Translation Protein Biosynthesis. Central Dogma DNA RNA protein transcription translation.
Aminoacyl tRNA Synthetases in Translation Aminoacylation of tRNA is a Two-step Reaction Long-range Communications in Bacterial Prolyl-tRNA Synthetases.
Water layer Protein Layer Copper center: QM Layer Computing Redox Potentials of Type-1 Copper Sites Using Combined Quantum Mechanical/Molecular Mechanical.
Section P THE GENETIC CODE AND tRNA
Gene expression. The information encoded in a gene is converted into a protein  The genetic information is made available to the cell Phases of gene.
PREDICTION OF CATALYTIC RESIDUES IN PROTEINS USING MACHINE-LEARNING TECHNIQUES Natalia V. Petrova (Ph.D. Student, Georgetown University, Biochemistry Department),
Translation.  Is the process in which mRNA provides a template for synthesis of polypeptide.
1 Protein synthesis How a nucleotide sequence is translated into amino acids.
BIOCHEMISTRY REVIEW Overview of Biomolecules Chapter 13 Protein Synthesis.
TRANSLATION In all things of nature there is something of the marvelous… (Aristotle) RNA-Directed Polypeptide Synthesis.
Purification and Enzymatic Activity of Cfd1 and Nbp35 Mierzhati Mushajiang, Eric Camire, and Deborah Perlstein Department of Chemistry, Boston University,
Exploring Cooperative Domain Dynamics in Thermus thermophilus Leucyl-tRNA Synthetase Using Low-frequency Normal Mode Calculations and Statistical Coupling.
Translation- Making the Protein
Protein Synthesis- the ”Stuff of Life“ Translation- Making the Protein.
Functions of RNA mRNA (messenger)- instructions protein
Exploring Electron Transfer-Induced Conformational Changes in NRH:Quinone Oxidoreductase Chee Yang, Alexander Jerome Greene, James C. Raucshnot, Jr., Sanchita.
Figure 5: Expression and solubility tests for constructs of CoVs. Coronaviruses are complex, positive-sense RNA viruses that cause mild to severe respiratory.
TOPIC 2.7 TRANSCRIPTION & TRANSLATION. Nucleus: the control center  contains nuclear envelope, nucleoli, chromatin, and distinct compartments rich in.
Protein Synthesis- the ”Stuff of Life“ Translation- Making the Protein.
Biochemistry: A Short Course Third Edition CHAPTER 39 The Genetic Code © 2015 W. H. Freeman and Company Tymoczko Berg Stryer.
Soybean Lipoxygenase: Which amino acids matter?
Experimental Kinetic Study to Explore the Impact of Macromolecular Crowding on Structure and Function of Escherichia coli Prolyl–tRNA Synthetase An Nam.
Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)
Reading the instructions and building a protein!
Relationship between Genotype and Phenotype
Natural Supramolecular Building Blocks
Volume 13, Issue 6, Pages (March 2004)
Finn Werner, Robert O.J Weinzierl  Molecular Cell 
Glen S. Cho, Jack W. Szostak  Chemistry & Biology 
Figure Number: 27-00CO Title: RNA Catalyst
Cell Protein Production
The Active Site of the Ribosome Is Composed of Two Layers of Conserved Nucleotides with Distinct Roles in Peptide Bond Formation and Peptide Release 
Kinetic Discrimination of tRNA Identity by the Conserved Motif 2 Loop of a Class II Aminoacyl-tRNA Synthetase  Ethan C. Guth, Christopher S. Francklyn 
Stephen Schuck, Arne Stenlund  Molecular Cell 
Volume 16, Issue 2, Pages (February 2008)
Volume 15, Issue 11, Pages (November 2008)
Hiroshi Murakami, Dimitrios Kourouklis, Hiroaki Suga 
Volume 19, Issue 2, Pages (July 2005)
Chapter 23 Using the Genetic Code.
A Shared Surface of TBP Directs RNA Polymerase II and III Transcription via Association with Different TFIIB Family Members  Xuemei Zhao, Laura Schramm,
Volume 1, Issue 2, Pages (January 1998)
Structure, Exchange Determinants, and Family-Wide Rab Specificity of the Tandem Helical Bundle and Vps9 Domains of Rabex-5  Anna Delprato, Eric Merithew,
ATPase Site Architecture and Helicase Mechanism of an Archaeal MCM
Claudia Schneider, James T. Anderson, David Tollervey  Molecular Cell 
Volume 11, Issue 2, Pages (February 2003)
Transfer RNA–Mediated Editing in Threonyl-tRNA Synthetase
George Simos, Anke Sauer, Franco Fasiolo, Eduard C Hurt  Molecular Cell 
Exchange of Regions between Bacterial Poly(A) Polymerase and the CCA-Adding Enzyme Generates Altered Specificities  Heike Betat, Christiane Rammelt, Georges.
Transcriptional Regulation by p53 through Intrinsic DNA/Chromatin Binding and Site- Directed Cofactor Recruitment  Joaquin M Espinosa, Beverly M Emerson 
Volume 8, Issue 9, Pages (April 1998)
Spb1p-Directed Formation of Gm2922 in the Ribosome Catalytic Center Occurs at a Late Processing Stage  Bruno Lapeyre, Suresh K. Purushothaman  Molecular.
Volume 1, Issue 4, Pages (March 1998)
Bacterial and Eukaryotic Phenylalanyl-tRNA Synthetases Catalyze Misaminoacylation of tRNAPhe with 3,4-Dihydroxy-L-Phenylalanine  Nina Moor, Liron Klipcan,
Volume 13, Issue 6, Pages (March 2004)
Molecular Mechanism of Drug-Dependent Ribosome Stalling
Presentation transcript:

Biochemical Studies to Probe the Domain-Domain Communication Pathways in E. coli Prolyl-tRNA Synthetase Heidi Schmit and Sanchita Hati Department of Chemistry, University of Wisconsin-Eau Claire, WI Biochemical Studies to Probe the Domain-Domain Communication Pathways in E. coli Prolyl-tRNA Synthetase Heidi Schmit and Sanchita Hati Department of Chemistry, University of Wisconsin-Eau Claire, WI Abstract Background Conclusions Methods Active-site titrations will be done to quantify the amount of the protein that is active and to find out the approximate catalytic activity of the wild- type and mutants of ProRS. ATP-PP i exchange assays will be performed for the WT and mutant proteins. This assay involves the first step in the two-step reaction, which is reversible, for the charging of tRNA. This assay is done without the presence of tRNA and the PP i is radioactively labeled so when the reactive goes in reverse direction, the ATP that is produced will become radioactively labeled. This gives an indirect approach to measure the activity of the ProRS. Aminoacylation assays will also be performed that will give the actual measurement of the catalytic activity of the wild-type and mutant enzymes. This assay has the presence of tRNA and is the second step of the aminoacylation two step reaction. Kinetic parameters of the WT and mutant enzymes will be compared to determine the impact of mutation on enzyme function. Double mutants will also be prepared and kinetic parameters will be determined. Overall, these assays will allow us to understand the role of each of the computationally predicted pathways in enzyme function. References- (1) Beuning et al. (2001) J. Biol. Chem. 276, (2) Crepin et al. (2006) Structure 14, (3) Hati et al. (2006) J. Biol. Chem. 281, (4) Johnson et al. (2013) Biochemistry, under revision. (5) Sanford et al. (2012) Biochemistry 51, Acknowledgements:  National Institute of Health (Grant number GM085779)  National Science Foundation through TeraGrid Resources  Office of Research & Sponsored Programs at UWEC  Learning and Technology Services at UWEC To further our understanding of the molecular mechanism of domain-domain communications in ProRS. Perform alanine-scanning mutagenesis to examine if all the predicted pathways of site-to-site communication are equally important for the efficient function of Ec ProRS. To explore the role of tRNA Pro in long-range communication in Ec ProRS. Prolyl-tRNA Synthetase Catalyzes the covalent attachment of proline to tRNA Pro in a two- step reaction: Misactivates alanine and possesses editing mechanism to hydrolyze Ala-tRNA Pro [1] Three domains [2]: Substrate-induced conformational change of catalytically important proline-binding loop (PBL) [2]. Overall activation efficiency (k cat /K M ) of the deletion mutant was decreased ~1200-fold relative to the wild-type enzyme [3] Ten of the twelve site-directed mutations were successful and the DNA sequence of all mutants are verified by DNA sequencing. Overexpression of the ten selected mutants has been completed. Currently, two of the mutants have been purified and concentrated and are ready for the last three steps of Scheme 1. Results Objectives Future Directions Pro + ATP + ProRS ⇆ Pro-AMP∙ProRS + PP i Pro-AMP∙ProRS + tRNA Pro → Pro-tRNA Pro + AMP + ProRS Aminoacyl-tRNA synthetases (AARSs) are a family of enzymes that catalyze the covalent attachment of amino acids to their corresponding transfer-RNA. These enzymes play critical roles in protein synthesis and viability. AARSs are comprised of many domains and each domain is responsible for carrying out a specific function for the proper attachment of correct amino acids to tRNAs. Previous research studies have shown that the protein dynamics, especially correlated motion between residue pairs, play an important role in domain-domain communication in these enzymes. Recently, using molecular simulations and bioinformatics, we have traced several potential pathways of residue-residue interactions through which correlated motions could be propagated from one site to another and thereby help coordinate functions of various domains. We are presently probing those computationally determined pathways through experimental mutational studies. We are specifically focused on one enzyme of this family - prolyl-tRNA synthetases (ProRS), which attaches proline on to the tRNA Pro. Currently, site-directed mutagenesis and kinetic studies are being carried out to study the role of specific mutations in the inter-domain communication and catalytic activity of this enzyme. Preliminary results of this study will be presented. Scheme 1 shows the steps that are involved in site-directed mutagenesis and kinetic studies. The blue boxes are the steps completed for ten of the twelve selected mutants or are in the process of being completed. The gold boxes are the future steps to be completed in the coming year. Aminoacyl-tRNA Synthetases in Translation Basic mechanism of tRNA aminoacylation: An amino acid and cognate tRNA are brought together to catalyze the formation of an ester bond in the active site using ATP. The charged tRNA is then transported to the ribosome for transcription into a protein. Catalaytic/Aminoacylation Domain: responsible for selecting the correct amino and catalyzing the attachment to the tRNA Editing Domain: Assures that the correct amino acid is attached to the tRNA Anticodon binding domain: responsible for recognizing the corresponding tRNA Kinetic parameters for amino acid activation by wild-type (WT) and mutants of E. coli ProRS a a Results are the average of 3 trials with the standard deviation indicated [4]. ΔΔG was calculated according to the equation ΔΔG = -RT ln (fold-decrease of k cat /K M ), where R is the gas constant, cal K -1 mol -1 and T is 310 K. ND indicates not detectable under the experimental conditions used. b Data is from reference [5]. Multiple Pathways of Domain-Domain Communication Representation of residue-residue interaction networks between the aminoacylation domain (R450/C443) and the editing domain (K279). The residues chosen for alanine scanning mutagenesis are shown in blue; residues shown in red are known to have considerable impact on enzyme function. T(h) Supernatant 2. Lysis Buffer 3. Wash Buffer mM Imidazole mM Imidazole Scheme 1. We have the following mutants sequenced and overexpressed: A197G, I404A, I414A, L281A, L304A, N232A, S207A, T199A, and V411A. Two mutants (A197G and L304A) have been purified and concentrated. The picture below on the left is an SDS PAGE gel showing overexpression of one of the mutant proteins using 0.1 M IPTG at 37  C. The second SDS PAGE gel on the right shows the final purified enzyme using affinity column chromatography. Each lane shows the progression of the protein purification. The single, prominent band in lane 5 is the successfully purified protein eluted with 100 mM imidazole.