Chapter 5 Macromolecular Assembly S E C T I O N II Copyright 2008 by Saunders/Elsevier. All rights reserved. Illustrations by Graham Johnson Cell Biology, 2e Thomas D. Pollard William C. Earnshaw with Jennifer Lippincott-Schwartz José A. Cardé-Serrano; PhD Biol 4018 – Celular Molecular Universidad de Puerto Rico - Aguadilla

Objetivos Explicar los conceptos importantes de los procesos de ensamblaje. Estudiar las ventajas evolutivas de los procesos de ensamblaje. Describir las regulaciones en los procesos de ensamblaje. Mencionar ejemplos de los procesos de ensamblaje.

Reorganization of microtubules between interphase and mitosis Green = microtubules; Blue = DNA; Red = centrosome; Pink = kinetochores Fig. 5-1 Copyright 2008 by Saunders/Elsevier. All rights reserved.

1. Large structures assemble from subunits 2. Specificity comes from multiple weak bonds on complementary surfaces 3. Subunits in symmetrical structures have identical or quasi-equivalent bonds between subunits Helices Icosahedrons 4. New properties emerge along assembly pathways 5. Regulation is imposed at multiple steps along pathways General principles of macromolecular assembly Copyright 2008 by Saunders/Elsevier. All rights reserved.

Conserves space in the genome; one small gene can encode all of the pieces of a large structure Allows synthesis of error-free components; errors in protein synthesis occur ~1/3000 residues, so most small subunits are free of errors Quality control: defective subunits can be discarded Subunits are easy to recycle Multiple modes of regulation are available Advantages of assembling large structures from small protein subunits Copyright 2008 by Saunders/Elsevier. All rights reserved.

Tobacco mosaic virus Fig From S. Dinesh-Kumar, Yale University -First virus assembled from purified protein and RNA proteinas/ 6300ncltds -1x106 ncltds si fuera un gen/proteinas -1 gen de 474 ncltds/2000 protinas EM Crystal structure Copyright 2008 by Saunders/Elsevier. All rights reserved.

1. Large structures assemble from subunits 2. Specificity comes from multiple weak bonds on complementary surfaces 3. Subunits in symmetrical structures have identical or quasi-equivalent bonds between subunits Helices Icosahedrons 4. New properties emerge along assembly pathways 5. Regulation is imposed at multiple steps along pathways General principles of macromolecular assembly Copyright 2008 by Saunders/Elsevier. All rights reserved.

~No covalent bonds between subunits! Hydrophobic effect usually predominates Hydrogen bonds, electrostatic bonds Subunits in macromolecular structures are held together by multiple weak bonds on complementary surfaces Fig. 5-2 Copyright 2008 by Saunders/Elsevier. All rights reserved.

Self-assembly of actin filaments Fig. 5-5 EM Copyright 2008 by Saunders/Elsevier. All rights reserved.

1. Large structures assemble from subunits 2. Specificity comes from multiple weak bonds on complementary surfaces 3. Subunits in symmetrical structures have identical or quasi-equivalent bonds between subunits Helices Icosahedrons 4. New properties emerge along assembly pathways 5. Regulation is imposed at multiple steps along pathways General principles of macromolecular assembly Copyright 2008 by Saunders/Elsevier. All rights reserved.

Nucleación – iniciacion de polimerizcion por actinas puras Concentración Crítica – concentracion de monomeros que determina si ocurre alargamiento o acortamiento

Polyoma SV40 Structure & assembly of DNA tumor viruses Fig Pentameric subunits interact at both 5-fold and 6-fold vertices by virtue of flexible C-terminal tails. Copyright 2008 by Saunders/Elsevier. All rights reserved.

Helices Icosahedrons Hexagonal arrays Symmetrical structures are constructed from identical subunits with identical or quasi-equivalent bonds Figs. 5-2 & 5-4 Copyright 2008 by Saunders/Elsevier. All rights reserved.

Simetría hexagonal Típicas de estructuras planas. Esta maximiza el contacto entre subunidades. – Cada subunidad esta rodeada por seis vecinos. –Membranas

Simetría helicoidal Comparable a una escalera circular. Cada subunidad esta localizada a una distancia fija del eje Y su ángulo de rotación es fijo con respecto a la subunidad anterior Poseen una o mas hebras Pueden ser sólidas (actina) o huecas (flagelos)

Simetría poligonal Estructura típica de macromoléculas cerradas Es favorecido por triángulos equivalentes Estos triángulos forman anillos de 5 o 6 triángulos.

Association of subunits brings together partial binding sites: formation of an actin trimer nucleus has all of the binding sites to grow an actin filament Conformational change creates a binding site: interactions with an actin filament & WASp cause a conformational change allowing Arp2/3 complex to grow an actin filament branch New properties emerge along assembly pathways Figs. 5-6 & Copyright 2008 by Saunders/Elsevier. All rights reserved.

1. Large structures assemble from subunits 2. Specificity comes from multiple weak bonds on complementary surfaces 3. Subunits in symmetrical structures have identical or quasi-equivalent bonds between subunits Helices Icosahedrons 4. New properties emerge along assembly pathways 5. Regulation is imposed at multiple steps along pathways General principles of macromolecular assembly Copyright 2008 by Saunders/Elsevier. All rights reserved.

Propiedades del mecanismo de ensamblaje Todos los procesos de ensamblaje dependen de la difusión y coalisión entre las subunidades. La razón de asociación depende de la concentración de las subunidades y de la k. –Limitada por la razón de difusión La razón de disociación determina cual complejo o macromolécula es lo suficientemente estable para participar en los mecanismos de ensamblaje.

Propiedades del mecanismo de ensamblaje La estabilidad de los complejos macromoleculares varia considerablemente debido a: –Constante de la razón de disociación –Los cambios conformacionale;s son en la mayoría seguidos de la formación de complejos entre subunidades. Las estructuras grandes usualmente se ensamblan utilizando mecanismos específicos en los cuales surgen nuevas propiedades. La iniciación del proceso de ensamblaje es menos favorecido que su propagación.

Regulación por biosíntesis y degradación de subunidades - Feedback negativo: concentración de subU de tubulina regula la estabilidad del mRNA de tubulina Regulación de nucleación - nucleación es menos favorable que alargamiento - MTOC – (centrosomas) Regulación por cambios en condiciones ambientales - pH, concentración de iones; - TMV – [Ca+2] ; uncoating del genoma RNA Regulación en puntos múltiples en las rutas Secuenciales de ensamblaje Copyright 2008 by Saunders/Elsevier. All rights reserved.

Regulación por modificaciones covalentes de las subunidades - fosforilaciones, defosforilaciones, proteólisis, hidroxilaciones, glucosilaciones, oxidaciones, metilaciones, etc… Regulación por proteínas accesorias - chaperonas - GroEL - andamiajes – p22 – guia el ensamblaje de a cápsula - nebulina – longitud de filamento de actina Regulación en puntos múltiples en las rutas Secuenciales de ensamblaje Copyright 2008 by Saunders/Elsevier. All rights reserved.

Association of subunits brings together partial binding sites: formation of an actin trimer nucleus has all of the binding sites to grow an actin filament Conformational change creates a binding site: interactions with an actin filament & WASp cause a conformational change allowing Arp2/3 complex to grow an actin filament branch New properties emerge along assembly pathways Figs. 5-6 & Copyright 2008 by Saunders/Elsevier. All rights reserved.

1. Large structures assemble from subunits 2. Specificity comes from multiple weak bonds on complementary surfaces 3. Subunits in symmetrical structures have identical or quasi-equivalent bonds between subunits Helices Icosahedrons 4. New properties emerge along assembly pathways 5. Regulation is imposed at multiple steps along pathways General principles of macromolecular assembly Copyright 2008 by Saunders/Elsevier. All rights reserved.

Structure of myosin-II filaments Fig. 5-7 Copyright 2008 by Saunders/Elsevier. All rights reserved. Muscle myosin thick filament Cytoplasmic myosin mini-filament EM

Assembly of cytoplasmic myosin-II mini-filaments Filaments assemble in three steps: dimers, tetramers and octamers. New properties emerge at each step. Fig. 5-8 Copyright 2008 by Saunders/Elsevier. All rights reserved.

Assembly of bacterial flagella Figs. 5-9 & 5-10 Flagella grow at the distal end by addition of flagellin subunits. At high flagellin concentrations elongation is limited by slow folding of part of the protein. Copyright 2008 by Saunders/Elsevier. All rights reserved.

Assembly of tobacco mosaic virus RNA allows elongation at neutral pH and determines the length Fig Copyright 2008 by Saunders/Elsevier. All rights reserved.

ATP ADP Bacteriophage T4 assembles from 49 gene products along three different pathways Fig Fig Copyright 2008 by Saunders/Elsevier. All rights reserved.