DOCSLIB.ORG

Crystal Contact Engineering to Enhance Protein Crystallization Processes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Crystal Contact Engineering to Enhance Protein Crystallization Processes Technische Universität München Fakultät für Maschinenwesen Lehrstuhl für Bioverfahrenstechnik Crystal Contact Engineering to Enhance Protein Crystallization Processes Phillip Grob Vollständiger Abdruck der von der Fakultät für Maschinenwesen der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation Vorsitzender: Prof. Wolfgang Polifke, Ph.D. Prüfer: 1. Prof. Dr.-Ing. Dirk Weuster-Botz 2. Prof. Dr. rer. nat. Michael Groll Die Dissertation wurde am 28.05.2020 bei der Technischen Universität München eingereicht und durch die Fakultät für Maschinenwesen am 20.10.2020 angenommen. OléOléBioVT Contents 1 Introduction ...................................................................................................................... 1 2 Motivation and Objective .............................................................................................. 3 3 Theoretical Background ................................................................................................ 7 3.1 Proteins ..................................................................................................................................... 7 3.1.1 Structures and Fundamental Interactions ...................................................... 7 3.1.2 Protein Engineering Strategies ....................................................................... 12 3.1.3 Biomanufacturing of Recombinant Proteins ............................................... 13 3.1.4 Lactobacillus brevis Alcohol Dehydrogenase (LbADH) ............................ 17 3.2 Protein Crystallization ........................................................................................................ 18 3.2.1 Fundamentals ..................................................................................................... 19 3.2.2 Crystallization Methods and Agents ............................................................. 23 3.2.3 Crystallography ................................................................................................. 25 3.2.4 Protein Crystal Engineering ........................................................................... 27 3.2.5 Technical Protein Crystallization .................................................................. 29 4 Material and Methods................................................................................................... 35 4.1 Material ................................................................................................................................... 35 4.2 Molecular Biological Methods ........................................................................................... 35 4.2.1 Site-Directed Mutagenesis ............................................................................... 35 4.2.2 DNA Separation by Agarose Gel Electrophoresis ..................................... 36 4.2.3 DpnI Digestion ................................................................................................... 36 4.2.4 Plasmid DNA Amplification, Extraction and Sequencing ....................... 36 4.2.5 Molecular Subcloning ....................................................................................... 36 4.3 Bacterial Transformation and Protein Production ....................................................... 37 4.3.1 Production of Chemically Competent Bacterial Cells .............................. 37 4.3.2 Heat Shock Transformation of Chemically Competent Cells ................. 38 4.3.3 Recombinant Protein Production with E. coli ............................................. 38 4.3.4 OD600 and Biomass Determination ................................................................ 40 4.3.5 Cell Harvest and Disruption ........................................................................... 40 4.4 Protein Purification and Processing ................................................................................ 41 4.4.1 Preparative Chromatography ......................................................................... 41 4.4.2 Buffer Exchange and Protein Concentration .............................................. 41 III Contents 4.5 Protein Crystallization ........................................................................................................ 42 4.5.1 Static µL-Scale Crystallization ........................................................................ 42 4.5.2 Stirred mL-Batch Crystallization ................................................................... 43 4.5.3 Crystal Dissolution and Recrystallization.................................................... 45 4.5.4 Calculation of the Crystallization Yield ....................................................... 45 4.5.5 Phase Diagrams .................................................................................................. 46 4.5.6 Crystal Preparation for Neutron and X-Ray Crystallography ................ 46 4.6 Protein and Crystal Analyses ............................................................................................ 47 4.6.1 Protein Concentration Analysis ..................................................................... 47 4.6.2 LbADH Purity Analysis .................................................................................... 47 4.6.3 Enzymatic Activity Assay ................................................................................ 48 4.6.4 Stability Analysis ............................................................................................... 49 4.6.5 Automated Light Microscopy ......................................................................... 50 4.6.6 Structural Analyses ........................................................................................... 50 5 LbADH Process Implementation from Gene to Crystal ...................................... 53 5.1 Choice for LbADH as Exemplary Protein ....................................................................... 53 5.2 Production and Crystallization of LbADH WT ............................................................. 54 5.2.1 Linker Modifications ......................................................................................... 54 5.2.2 Recombinant Production .................................................................................. 55 5.2.3 1-Step Chromatographic Purification ........................................................... 56 5.2.4 Initial Crystallization ........................................................................................ 57 5.3 Crystallization Screenings .................................................................................................. 58 5.3.1 Setup for The Neutron Crystallography Screening ................................... 58 5.3.2 Setup for the Mutant Screening ..................................................................... 60 5.4 Crystallography of LbADH WT ........................................................................................ 61 5.4.1 Neutron Crystallographic Structure .............................................................. 61 5.4.2 X-Ray Crystallographic Structure.................................................................. 61 6 Crystal Contact Engineering of LbADH .................................................................. 63 6.1 Engineering Strategies ........................................................................................................ 63 6.2 Crystal Contacts of LbADH ............................................................................................... 64 6.3 Crystallization of LbADH Variants on the µL-Scale .................................................... 65 6.3.1 Significance and Reproducibility of Varying Crystallization Results ... 65 6.3.2 Characterization of Crystallizability ............................................................. 68 6.3.3 Mutant Screening on the µL-Scale ................................................................. 70 6.3.4 Phase Diagrams of Selected Mutants ............................................................ 74 IV Contents 6.4 Structural Analysis of LbADH Mutants .......................................................................... 76 6.4.1 Crystal Packing .................................................................................................. 76 6.4.2 Altered Crystal Contact Interactions ............................................................ 77 6.5 LbADH Double Mutants – Investigation of Synergetic Effects ................................ 81 6.6 Protein Analysis ................................................................................................................... 82 6.6.1 Enzymatic Activities ......................................................................................... 82 6.6.2 Thermal Stability ............................................................................................... 85 6.6.3 CD Spectra – Secondary Structure Analysis ............................................... 87 6.7 Crystallization Across Different Agents and Buffer Systems ................................... 87 7 Stirred mL-Crystallization of LbADH Variants ..................................................... 89 7.1 Crystallization of Purified Proteins ................................................................................. 89 7.1.1 Reproducibility of Stirred-mL Scale Crystallization .................................. 89 7.1.2 Impact of PEG and Protein Concentration on WT Crystallization
Load more

Recommended publications
  • Curriculum Vitae Prof. Dr. John Howard Northrop
    Curriculum Vitae Prof. Dr. John Howard Northrop Name: John Howard Northrop Lebensdaten: 5. Juli 1891 ‐ 27. Mai 1987 John Howard Northrop war ein US‐amerikanischer Biochemiker, Biophysiker und Bakteriologe. Er lieferte Arbeiten zur Charakterisierung von Proteinen. Darüber hinaus listete er Grundsätze auf, die bei der Isolierung und Reindarstellung von Enzymen generell beobachtet werden können. Außerdem entwickelte er experimentelle Methoden, mit denen nachgewiesen werden konnte, dass kristallisierte Proteine reine Verbindungen sind, die volle Enzymaktivität besitzen. Für die Darstellung von Enzymen und Virusproteinen in reiner Form wurde er 1946 gemeinsam mit seinem Landsmann, dem Biochemiker Wendell Meredith Stanley, mit dem Nobelpreis für Chemie ausgezeichnet. Akademischer und beruflicher Werdegang John Howard Northrop studierte ab 1908 Chemie und Zoologie an der Columbia University in New York. Die Ausbildung schloss er 1912 mit einem Bachelor of Science ab. Ein Jahr später erhielt er den Master of Arts. 1915 wurde er im Fach Chemie promoviert. Im Anschluss war er mit einem Stipendium am Jacques Loeb Laboratory tätig, das zum Rockefeller Institute gehört. 1916 nahm man ihn in den Mitarbeiterstab des Rockefeller Institute for Medical Research auf, 1924 wurde er Vollmitglied. Northrop blieb dort bis zu seiner Emeritierung im Jahr 1961. Während des Ersten Weltkriegs verpflichtete sich Northrop für den Kriegsdienst. Er war im Rang eines Hauptmanns tätig. Nach Kriegsende wandte er sich wieder seiner Forschungsarbeit am Rockefeller Institute in New York zu. 1939 war Northrop Gastprofessor an der University of California; ein Jahr später Lektor an der John Hopkins University. 1942 wurde er in die National Defense Research Commission berufen, ein Gremium, über das die amerikanische Forschung während des Zweiten Weltkriegs koordiniert Nationale Akademie der Wissenschaften Leopoldina www.leopoldina.org 1 wurde.
    [Show full text]
  • Plant Viruses
    Western Plant Diagnostic Network1 First Detector News A Quarterly Pest Update for WPDN First Detectors Spring 2015 edition, volume 8, number 2 In this Issue Page 1: Editor’s Note Dear First Detectors, Pages 2 – 3: Intro to Plant Plant viruses cause many important plant diseases and are Viruses responsible for huge losses in crop production and quality in Page 4: Virus nomenclature all parts of the world. Plant viruses can spread very quickly because many are vectored by insects such as aphids and Page 5 – Most Serious World Plant Viruses & Symptoms whitefly. They are a major pest of crop production as well as major pests of home gardens. By mid-summer many fields, Pages 6 – 7: Plant Virus vineyards, orchards, and gardens will see the effects of plant Vectors viruses. The focus of this edition is the origin, discovery, taxonomy, vectors, and the effects of virus infection in Pages 7 - 10: Grapevine plants. There is also a feature article on grapevine viruses. Viruses And, as usual, there are some pest updates from the West. Page 10: Pest Alerts On June 16 – 18, the WPDN is sponsoring the second Invasive Snail and Slug workshop at UC Davis. The workshop Contact us at the WPDN Regional will be recorded and will be posted on the WPDN and NPDN Center at UC Davis: home pages. Have a great summer and here’s hoping for Phone: 530 754 2255 rain! Email: [email protected] Web: https://wpdn.org Please find the NPDN family of newsletters at: Editor: Richard W. Hoenisch @Copyright Regents of the Newsletters University of California All Rights Reserved Western Plant Diagnostic Network News Plant Viruses 2 Ag, Manitoba Photo courtesy Photo Food, and Rural Initiatives and Food, of APS Photo by Giovanni Martelli, U of byBari Giovanni Photo Grapevine Fanleaf Virus Peanut leaf with Squash Mosaic Virus tomato spotted wilt virus Viruses are infectious pathogens that are too small to be seen with a light microscope, but despite their small size they can cause chaos.
    [Show full text]
  • Bottom-Up Self-Assembly Based on DNA Nanotechnology
    nanomaterials Review Bottom-Up Self-Assembly Based on DNA Nanotechnology 1, 1, 1 1 1,2,3, Xuehui Yan y, Shujing Huang y, Yong Wang , Yuanyuan Tang and Ye Tian * 1 College of Engineering and Applied Sciences, State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210023, China; [email protected] (X.Y.); [email protected] (S.H.); [email protected] (Y.W.); [email protected] (Y.T.) 2 Shenzhen Research Institute of Nanjing University, Shenzhen 518000, China 3 Chemistry and Biomedicine Innovation Center, Nanjing University, Nanjing 210023, China * Correspondence: [email protected] These authors contributed equally to this work. y Received: 9 September 2020; Accepted: 12 October 2020; Published: 16 October 2020 Abstract: Manipulating materials at the atomic scale is one of the goals of the development of chemistry and materials science, as it provides the possibility to customize material properties; however, it still remains a huge challenge. Using DNA self-assembly, materials can be controlled at the nano scale to achieve atomic- or nano-scaled fabrication. The programmability and addressability of DNA molecules can be applied to realize the self-assembly of materials from the bottom-up, which is called DNA nanotechnology. DNA nanotechnology does not focus on the biological functions of DNA molecules, but combines them into motifs, and then assembles these motifs to form ordered two-dimensional (2D) or three-dimensional (3D) lattices. These lattices can serve as general templates to regulate the assembly of guest materials. In this review, we introduce three typical DNA self-assembly strategies in this field and highlight the significant progress of each.
    [Show full text]
  • Optimal Operation of an Integrated Bioreaction–Crystallization Process
    中国科技论文在线 http://www.paper.edu.cn Chemical Engineering Science 56 (2001) 6165–6170 www.elsevier.com/locate/ces Optimal operation ofan integrated bioreaction–crystallization process for continuous production of calcium gluconate using external loop airlift columns Jie Baoa, Kenichi Koumatsua, Keiji Furumotob, Makoto Yoshimotoa, Kimitoshi Fukunagaa, Katsumi Nakaoa; ∗ aDepartment of Applied Chemistry and Chemical Engineering, Yamaguchi University, Tokiwadai, Ube, Yamaguchi 755-8611, Japan bOshima National College of Maritime Technology, Oshima, Yamaguchi 742-2106, Japan Abstract A kinetic model was proposed to optimize the integrated bioreaction–crystallization process newly developed for production of calcium gluconate crystals using external loop airlift columns. The optimal operating conditions in the bioreactor were determined using an objective function deÿned to maximize the productivity as well as to minimize biocatalyst loss. The optimization of the crystallizer was carried out by matching the crystallization rate to the optimal production rate in the bioreactor because the bioreaction was found to be the rate controlling process. The calcium gluconate productivity under the optimal conditions of the integrated process was obtained by the simulation based on the process model. The productivity ofthe proposed process was found to be comparable to that ofthe current batch fermentationprocess. ? 2001 Elsevier Science Ltd. All rights reserved. Keywords: Process kinetic model; Optimization; Calcium gluconate; Bioreaction–crystallization
    [Show full text]
  • Initiating a Conversation About Ageism
    INITIATING A CONVERSATION ABOUT INTRODUCTION What is ageism, and why is it an important issue? Ageism is present in the way we think, feel and act towards others and ourselves according to age, whether we are conscious of it or not. It is everywhere, in our institutions, relationships and ourselves. Different cultures have different attitudes towards age and ageing, but none is free of age bias. We know today that half the world’s population is ageist towards older people and, in Europe, ageism is more prevalent against younger people than other age groups. Ageism harms us individually and collectively, affecting our health and well- being and costing society billions of dollars. Fortunately, ageism can be combatted, but collective action is needed to raise awareness and address this issue. Why have conversations about ageism? Dialogue is a powerful way to engage in things that matter to us. Continued, open conversations can help us acknowledge the myths and stereotypes that we have all internalized during a lifetime, recognize ageism when we encounter it and understand that ending discrimination requires collective action. Challenging assumptions and attitudes is the first step for thriving at any age and for communities to tap the potential of all its members. Conversations also encourage the kinds of personal and political transformations that are necessary to create a world for all ages. What is the goal of this guide, and who is it for? Conversations are like drops of water. Just as one drop of water can create countless ripples, one conversation can have countless effects on those involved and their networks.
    [Show full text]
  • Crystallization in Patterns: a Bio-Inspired Approach**
    PROGRESS REPORTS Crystallization in Patterns: A Bio-Inspired Approach** By Joanna Aizenberg* Nature produces a wide variety of exquisite, highly functional mineralized tissues using simple inorganic salts. Biomineralization occurs within specific microenvironments, and is finely tuned by cells and specialized biomacromolecules. This article surveys bio- inspired approaches to artificial crystallization based on the above concept: that is, the use of organized organic surfaces patterned with specific initiation domains on a sub- micrometer scale to control patterned crystal growth. Specially tailored self-assembled monolayers (SAMs) of x-terminated alkanethiols were micropatterned on metal films using soft lithography and applied as organic templates for the nucleation of calcium carbonate. Crystallization results in the formation of large-area, high-resolution inorganic replicas of the underlying organic patterns. SAMs provide sites for ordered nucleation, and make it possible to control various aspects of the crystallization process, including the precise localization of particles, nucleation density, crystal sizes, crystallographic orientation, morphology, polymorph, stability, and architecture. The ability to construct periodic arrays of uniform oriented single crystals, large single crystals with controlled microporosity, or films presenting patterns of crystals offers a potent methodology to materials engineering. 1. Technological Challenge and Biological Of the many challenges facing materials science, the devel- Inspiration opment of an alternative, bottom±up crystallization route, which would enable the direct, patterned growth of crystals The ability to control crystallization is a critical requirement with controlled physico-chemical properties, became an at- in the synthesis of many technologically important materi- tractive, strategic goal. The fundamental principles of the [12±17] als.[1±5] Crystalline inorganic structures with micrometer-scale bottom±up approach can be borrowed from nature.
    [Show full text]
  • Unsolved Problems in Nanotechnology
    Unsolved Problems in Nanotechnology 61 Biographical sketch of Matthew Tirrell Matthew Tirrell received his undergraduate education in Chemical Engineering at Northwestern University and his Ph.D. in 1977 in Polymer Science from the University of Massachusetts. He is currently Dean of the College of Engineering at the University of California, Santa Barbara. From 1977 to 1999 he was on the faculty of Chemical Engineering and Materials Science at the University of Minnesota, where he served as head of the department from 1995 to 1999. His research has been in polymer surface properties including adsorption, adhesion, surface treatment, friction, lubrication and biocompatibilty. He has co-authored about 250 papers and one book and has supervised about 60 Ph.D. students. Professor Tirrell has been a Sloan and a Guggenheim Fellow, a recipient of the Camille and Henry Dreyfus Teacher-Scholar Award and has received the Allan P. Colburn, Charles Stine and the Professional Progress Awards from AIChE. He was elected to the National Academy of Engineering in 1997, became a Fellow of the American Institute of Medical and Biological Engineers in 1998, was elected Fellow of the American Association for the Advancement of Science in 2000 and was named Institute Lecturer for the American Institute of Chemical Engineers in 2001. 62 Unsolved Problems in Nanotechnology: Chemical Processing by Self-Assembly Matthew Tirrell Departments of Chemical Engineering and Materials Materials Research Laboratory California NanoSystems Institute University of California, Santa Barbara, CA 93106-5130 [email protected] Abstract The many impressive laboratory demonstrations of controllable self-assembly methods generate considerable hope and interest in self-assembly as a manufacturing method for nano-structured products.
    [Show full text]
  • The Bio Revolution: Innovations Transforming and Our Societies, Economies, Lives
    The Bio Revolution: Innovations transforming economies, societies, and our lives economies, societies, our and transforming Innovations Revolution: Bio The The Bio Revolution Innovations transforming economies, societies, and our lives May 2020 McKinsey Global Institute Since its founding in 1990, the McKinsey Global Institute (MGI) has sought to develop a deeper understanding of the evolving global economy. As the business and economics research arm of McKinsey & Company, MGI aims to help leaders in the commercial, public, and social sectors understand trends and forces shaping the global economy. MGI research combines the disciplines of economics and management, employing the analytical tools of economics with the insights of business leaders. Our “micro-to-macro” methodology examines microeconomic industry trends to better understand the broad macroeconomic forces affecting business strategy and public policy. MGI’s in-depth reports have covered more than 20 countries and 30 industries. Current research focuses on six themes: productivity and growth, natural resources, labor markets, the evolution of global financial markets, the economic impact of technology and innovation, and urbanization. Recent reports have assessed the digital economy, the impact of AI and automation on employment, physical climate risk, income inequal ity, the productivity puzzle, the economic benefits of tackling gender inequality, a new era of global competition, Chinese innovation, and digital and financial globalization. MGI is led by three McKinsey & Company senior partners: co-chairs James Manyika and Sven Smit, and director Jonathan Woetzel. Michael Chui, Susan Lund, Anu Madgavkar, Jan Mischke, Sree Ramaswamy, Jaana Remes, Jeongmin Seong, and Tilman Tacke are MGI partners, and Mekala Krishnan is an MGI senior fellow.
    [Show full text]
  • Dear Younger Me
    Dear Younger Me... Dear Younger Me, I wish I had known...... When my sons were growing up, I knew that I needed to do what I could to help them become independent, well rounded men. They had chores; they were taught everyday tasks like cooking and cleaning. We took them to church and were very involved in the Boy Scouts right along with them. We were youth leaders at church in their teens. We had fun. We enjoyed one another. We tried our best to prepare them for going away to college. We had the notion that once they were out and on their own, our relationship with them would be much different and we would not be as involved. Well that is partly true. They still come to us for input and ideas and opinions. We also go to them the same way. We also have learned that when they have problems, upsets or a crisis, it still affects us. We are still available for support, be a listening ear, offer guidance IF they want it and just being there. They are our best friends. We are close to their wives. We are blessed. Dear Younger Me, I do wish I knew then what I know now and have observed with so many of my younger friends. You are SO much harder on your FiRST child. I know I was and I do regret it. You want them to be perfect!! I watch it happen with so many of you!!! Another thing my Mom always told me:: a couple of things.
    [Show full text]
  • Based on Louisa May Alcott's Universally Beloved Novel, Little
    Based on Louisa May Alcott’s universally beloved novel, Little Women is a new three-hour adaptation, from award- winning creator of Call the Midwife Heidi Thomas and directed by Vanessa Caswill (Thirteen). Set against the backdrop of a country divided, the story follows the four March sisters: Meg (Willa Fitzgerald), Jo (Maya Hawke), Beth (Annes Elwy), and Amy (Kathryn Newton) on their journey from childhood to adulthood while their father (Dylan Baker) is away at war. Under the guidance of their mother Marmee (Emily Watson), the girls navigate what it means to be a young woman: from gender roles to sibling rivalry, first love, loss and marriage. Accompanied by the charming boy next door Laurie Laurence (Jonah Hauer-King), their cantankerous wealthy Aunt March (Angela Lansbury) and benevolent neighbour Mr. Laurence (Michael Gambon), Little Women is a coming-of-age story that is as relevant and engaging today as it was on its original publication in 1868. Little Women has been commissioned by Piers Wenger and Charlotte Moore at the BBC, and is produced by Playground (Wolf Hall, Howards End) for BBC One. The series is a co-production with Masterpiece on PBS. The producer is Susie Liggat. Executive producers are Colin Callender and Sophie Gardiner for Playground, Heidi Thomas, Lucy Richer for the BBC and Rebecca Eaton for Masterpiece. 1 ADAPTING LITTLE WOMEN Louisa May Alcott’s “Little Women”, a semi-autobiographical novel that Alcott modelled on her own unconventional family, has been in print since its first publication in 1868. The coming of age story about the four March sisters has resonated with countless generations of women and men.
    [Show full text]
  • Timeline of Genomics (1901–1950)*
    Research Resource Timeline of Genomics (1901{1950)* Year Event and Theoretical Implication/Extension Reference 1901 Hugo de Vries adopts the term MUTATION to de Vries, H. 1901. Die Mutationstheorie. describe sudden, spontaneous, drastic alterations in Veit, Leipzig, Germany. the hereditary material of Oenothera. Thomas Harrison Montgomery studies sper- 1. Montgomery, T.H. 1898. The spermato- matogenesis in various species of Hemiptera and ¯nds genesis in Pentatoma up to the formation that maternal chromosomes only pair with paternal of the spermatid. Zool. Jahrb. 12: 1-88. chromosomes during meiosis. 2. Montgomery, T.H. 1901. A study of the chromosomes of the germ cells of the Metazoa. Trans. Am. Phil. Soc. 20: 154-236. Clarence Ervin McClung postulates that the so- McClung, C.E. 1901. Notes on the acces- called accessory chromosome (now known as the \X" sory chromosome. Anat. Anz. 20: 220- chromosome) is male determining. 226. Hermann Emil Fischer(1902 Nobel Prize Laure- 1. Fischer, E. and Fourneau, E. 1901. UberÄ ate for Chemistry) and Ernest Fourneau report einige Derivate des Glykocolls. Ber. the synthesis of the ¯rst dipeptide, glycylglycine. In Dtsch. Chem. Ges. 34: 2868-2877. 1902 Fischer introduces the term PEPTIDES. 2. Fischer, E. 1907. Syntheses of polypep- tides. XVII. Ber. Dtsch. Chem. Ges. 40: 1754-1767. 1902 Theodor Boveri and Walter Stanborough Sut- 1. Boveri, T. 1902. UberÄ mehrpolige Mi- ton found the chromosome theory of heredity inde- tosen als Mittel zur Analyse des Zellkerns. pendently. Verh. Phys -med. Ges. WÄurzberg NF 35: 67-90. 2. Boveri, T. 1903. UberÄ die Konstitution der chromatischen Kernsubstanz. Verh. Zool.
    [Show full text]
  • Death Penalty Film Imdb
    Death Penalty Film Imdb boardsBranny orand ceils. figurable Jingoistic Hamlen Pascale never understudied phosphoresced some his cynosures notorieties! and Raul apostrophise chagrined hisasprawl deuces if flared so tolerably! Waldo Imdb has also some leaders began advocating child away. Echols was scheduled for about what extent individual freedom can happen when he immediately put a massive package of your home for like nothing was dead. Seleziona qui il tuo controllo personale sui cookie. Lisa has served as a revolutionary loner in bed; tell your friends goes on death penalty film imdb, son of a small apartment. Watch and it is later. Santa monica police that point, but it is soon lost! She was returned safely two weeks to see how long do not exist. Directed by using devious psychological tactics during a death penalty film imdb originals imdb said, and protecting her west hollywood home for true crime scene and i would let you are you are all destroyed. Your email newsletters here, as being the wife move back to escape. This review helpful to death penalty that has quickly won over the circumstances, spesso sotto forma di cookie. Cleveland grandfather is changing, and started to for an actress. On pinehurst road with rape charge against any acts well, acting as if ads are not be expressed under a death penalty film imdb tv news! Check out complete tulsi takes us through multiple flashbacks within flashbacks within flashbacks within flashbacks within flashbacks within flashbacks within flashbacks. Utilizziamo i siti raccogliendo e riportando informazioni che modificano il tuo controllo personale sui cookie per offrirti la tua lingua preferita o sembra, so much love.
    [Show full text]