Green Fluorescent Proteins

Methods in Cell Biology VOLUME 58 Green Fluorescent Proteins Series Editors Leslie Wilson Department of Biological Sciences University of Cahfomia, Santa Barbara Santa Barbara, California Paul Matsudaira Whitehead Institute for Biomedlcd Research and Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts Methods in Cell Biology Prepared under the Auspices of the American Society for Cell Biology VOLUME 58 Green Fluorescent Proteins Edited by Kevin F. Sullivan Department of Cell Biology The Scripps Research Institute La Jolla, California Steve A. Kay Department of Cell Biology The Scripps Research Institute La Jolla, California ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto Cover photo credit (paperback edition only): Mitosis in human cells is revealed by fluorescence microscopy showing GFP-labeled chromosomes that have incorporated a histone H2B-GFP fusion protein (green) coupled with immunofluorescence imaging of microtubules (blue) and centromeres (orange). The image was acquired using a Bio-Rad MRC-1024 confocal microscope. Provided by Kevin F. Sullivan. This book is printed on acid-free paper. @ Copyright 0 1999 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923). for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1999 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0091-679X/99 $25.00 Academic Press a division of Harcourr Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com Academic Press Limited 24-28 Oval Road, London NWl 7DX, UK http://www.hbuk.co.uk/ap/ International Standard Book Number: 0-12-544160-6 (case) International Standard Book Number: 0-12-676075-6 (pb) PRINTED IN THE UNlTEDSTATESOF Ah4ERICA 9 8 9 9 0 0 0 1 0 2 0 3 E B 9 8 7 6 5 4 3 2 1 CONTENTS Contributors xi Preface xv 1. Biophysics of the Green Fluorescent Protein F. G . Prendergast I. Introduction 11. Protein Folding and the Generation of This Chromophore 111. The Biophysics of the Fluorescence of GTP IV. Resonance Energy Transfer Involving GFP V. Summary References 1 3 8 15 16 17 2. Understanding Structure-Function Relationships in the Aequorea victoria Green Fluorescent Protein Andrew B. Cubitt, Leslie A . Woollenweber, and Roger Heim I. Introduction 11. Structure 111. IV. V. VI. Chromophore Formation Effects of Mutations on the Spectroscopic Properties of GFP Effects of Mutations That Improve Thermosensitivity The Development of Enhanced Mutants References 19 20 21 22 26 27 29 3. Quantitative Imaging of the Green Fluorescent Protein (GFP) David W. Piston, George H . Patterson, and Susan M . Knobel I. Introduction 3