Table Of Content

Photochemical Probes in Biochemistry NATO ASI Series Advanced Science Institutes Series A Series presenting the results of activities sponsored by the NA TO Science Committee, which aims at the dissemination of advanced scientific and technological knowledge, with a view to strengthening links between scientific communities. The Series is published by an international board of publishers in conjunction with the NATO Scientific Affairs Division A Life Sciences Plenum Publishing Corporation B Physics London and New York C Mathematical Kluwer Academic Publishers and Physical Sciences Dordrecht, Boston and London D Behavioural and Social Sciences E Applied Sciences F Computer and Systems Sciences Springer-Verlag G Ecological Sciences Berlin, Heidelberg, New York, London, H Cell Biology Paris and Tokyo Series C: Mathematical and Physical Sciences· Vol. 272 Photochemical Probes in Biochemistry edited by Peter E. Nielsen Department of Biochemistry B, The Panum Institute, University of Copenhagen, Copenhagen, Denmark , • Kluwer Academic Publishers Dordrecht / Boston / London Published in cooperation with NATO Scientific Affairs Division Proceedings of the NATO Advanced Research Workshop on Photochemical Probes in Biochemistry Copenhagen, Denmark 14-19 August 1988 Library of Congress Cataloging in Publication Data NATO Advanced Research Horkshop on Photoche~lcal Probes In Bloche~lstry (1988 : Copenhagen. Denmark) Photoche~lcal probes In biochemistry: proceedings of the NATO Advanced Research Horkshop on Photochemical Probes In Bloche~lstry. Copenhagen. Den~ark. August 14-19. 1988 I edited by Peter E. Nielsen. p. c~. -- (NATO ASI series. Series C. MatheMatical and physical sciences; vol. 272) Includes Indexes. ISBN -13: 978-94-010-6905-2 e-ISBN-13: 978-94-009-0925-0 DOl: 10.1007/978-94-009-0925-0 1. Photoafflnity labellng--Congresses. 2. Biochemlstry -Congresses. I. Nielsen. Peter E. • 1951- • II. Title. III. Series: NATO ASI series. Series C. Mathematical and physical sciences : no. 272. QP519.9.P48N37 1988 574. 19' 285--dc 19 89-31038 CIP ISBN -13: 978-94-010-6905-2 Published by Kluwer Academic Publishers, P.O. Box 17,3300 AA Dordrecht, The Netherlands. Kluwer Academic Publishers incorporates the publishing programmes of D. Reidel, Martinus Nijhoff, Dr W. Junk and MTP Press. Sold and distributed in the U.S.A. and Canada by Kluwer Academic Publishers, 101 Philip Drive, Norwell, MA 02061, U.S.A. In all other countries, sold and distributed by Kluwer Academic Publishers Group, P.O. Box 322, 3300 AH Dordrecht, The Netherlands. printed on acid free paper All Rights Reserved © 1989 by Kluwer Academic Publishers Softcover reprint of the hardcover 1st edition 1989 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photo copying, recording or by any information storage and retrieval system, without written permission from the copyright owner. T ABLE OF CONTENTS Preface ix 1. Photolabeling Reagent Design O. Buchardt, U. Hendriksen, T. Koch and P.E. Nielsen 2. Nucleophilic Aromatic Photo Substitutions on Nitrophenyl Ethers. A New Photoaffinity Labelling Technique J. Marquet and M. Moreno-Manas 11 3. Photochemistry of Aryl and Aroyl Azides: Application to Photo labeling of Biological Systems G.B. Schuster 31 4. Membrane Protein Labelling with Photoreactive Phospholipid Analogues C. Montecucco, G. Schiavo, E. Papini, M. Tomasi and R. Bisson 43 5. 125J_ASA_PE, A Photoactivable, Radioactive Phospholipid-Analogue, Designed for Photolabeling Proteins in Contact with Phospholipid Head Groups. Selective Photolabeling of Subunits a in Thermo- philic A TPsynthase TF O·F 1 Z. Gao and E. Bauerlein 59 6. Photoaffinity Labelling of Receptors of Melanocyte-Stimulating Hormone A.N. Eberle, F. Solca, W. Siegrist, T. Scimonelli, J. Girard and P.N.E. de Graan 67 7. Topographical Analysis of the Torpedo Marmorata Acetylcholine Receptor by Energy Transfer Photoaffinity Labeling Using Aryl diazonium Derivatives F. Kotzyba-Hibert, A. Jaganathen, J. Langenbuch-Cachat, M. Goeldner, C. Hirth, M. DenniS, J. Giraudat, J.L. Galzi, C. Mulle, J.P. Changeux, C. Bon, J.Y. Chang, C. Lazure and M. Chretien 85 8. Photosuicide Labelling L. Ehret-Sabatier, B. Kieffer, M.P. Goeldner and C.G. Hirth 107 9. Antibiotic Photoaffinity Labeling Probes of Escherichia coli Ribosomal Structure and Function B.S. Cooperman, M.A. Buck, C.L. Fernandez, C.J. Weitz mann and B.F.D. Ghrist 123 vi 10. Dissection of the Ribosomal Peptidyl Transferase Center by Photo affinity Labeling: P- and A-sites are Located at Different Posi tions in Domain V of 23 S RNA: E. Kuechler, G. Steiner and A. Barta 141 11. Studying the Cytoskeleton by Label Transfer Crosslinking: Uses and Limitations M.A. Schwartz 157 12. RecA-Directed Hybridization of Psoralen-Monoadducted DNA Oligonucleotides to Duplex Targets S. Cheng, H.B. Gamper and J.E. Hearst 169 13. The Use of Psoralen-Photocrosslinking for the Analysis of the Chromatin Structure during Transcription J.M. Sogo, A. Conconi and R.M. Widmer 179 14. Metal Complexes as Photochemical Probes of DNA Structure J.K. Barton 195 15. Laser-Induced Strand Break Formation of Polyuridylic Acid in the Presence and Absence of Tris (2,2'-BipyridyI) Ruthenium Chloride and K2S208 D. Schulte-Frohlinde, A.B. Tossi and H. Gorner 199 16. Photonucleases 0. Buchardt, G. Karup, M. Egholm, T. Koch, U. Henriksen, M. Meldal, C. Jeppesen and P.E. Nielsen 209 17. Sequence-Targeted Photochemical Reactions in Single-Stranded and Double-Stranded Nucleic Acids by Oligonucleotide-Photosensitizer Conjugates C. Helene, T. Le Doan and N.T. Thuong 219 18. Photofootprinting Analysis of Protein-DNA Interactions P.E. Nielsen and C. Jeppesen 231 19. Studies of the Binding and Biological Actions of Ethidium K.L. Yielding 241 20. Quantitative Photoaffinity Labeling of Escherichia coli RNA Polymerase Transcription Complexes by Nascent RNA T.M. Stackhouse and C.F. Meares 261 21. "Caged" Compounds to Probe the Dynamics of Cellular Processes: Synthesis and Properties of some Novel Photosensitive P-2-Nitro benzyl Esters of Nucleotides J.F. Wootton and D.R. Trentham 277 vii General Comments on the Use of Photochemical Probes in Biochem istry. Advantages, Problems and Pitfalls 297 List of Participants 299 Author Index 303 Keyword Index 305 PREFACE The concept of using photochemical probes in the study of biological systems was developed by Westheimer who published the first photoaffinity labeling experiments more than twenty years ago (J.Bio1. Chem. 237, (1962) 3006). Since then the concept has been used successfully in various areas of biochemistry and recently several new interesting and exciting aspects of the concept have been developed. It is the general opinion by scientists in the "field" that the full potential of photochemical probes in biochemical studies has far from been exploited yet. This is mostly due to the interdisciplinary character of the concept involving photochemistry, synthetic chemistry as well as biochemistry/molecular biology. The perspective of the NATO advanced workshop on "Photochemical Probes in Biochemistry", held in Holte (Copenhagen) Denmark 14-19, August, 1988, was several fold. The workshop was to give an account of the "state of the art" of using photochemical probes in biochemistry as well as to bring together specialists in photochemistry, syn thetic chemistry and molecular biology in order to analyze advantages as well as the inherent problems and pitfalls of the concept and provide suggestions and guidelines for fu ture research. Furthermore, it is the hope of the editor that the present publication which gives an account of the lectures presented at the workshop, will provide an introduction to scientists who are not familiar with photochemical probes, but to whom these could help answer central and pertinent questions. I hope this book will serve as a source of general infor mation about photochemical probes in biochemistry, and an entrance to the relevant original literature as well as a source of inspiration for novel applications and experimen tal designs. This workshop and the present publication was generously supported by NATO Division of Scientific Affairs. I also want to thank the participants of the workshop and the con tributors to this publication for their enthusiasm and cooperation. Support from Boehringer Mannheim and Amersham, Denmark, is also acknowledged. Peter E. Nielsen August, 1988 PHOTOLABELXNG REAGENT DESXGN Ole Buchardt, Ulla Henriksen, Troels Koch, and Peter E. Nielsen (a) Research Center for Medical Biotechnology, The H. C. 0rsted Institute, University of Copenha gen, Universi tetsparken 5, DK-2l00 Copenhagen 0, Denmark; (a) Department of Biochemistry B, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark ABSTRACT. Photolabeling reagents are composed of some or all of the following ligands: Photoprobes, thermal probes, labels, linkers, and cleavable sites. The design and func tion of such ligands are discussed on the basis of our own attempts to design and test novel ligands and re agents. Novel ligands include diazocyclopentadienylcar bonyloxy photoprobes, biotinyl labels and selenium con taining linkers which can be cleaved by very mild oxi dation. XNTRODUCTXON The use of photochemically activated reagents in biology and medicine has steadily gained importance, and consider ing their wealth of possibilities we can expect to see further increase in the range and application of photo chemistry as a tool in these areas. They can be divided into (i) photolabeling, (ii) photoaffinity labeling, and (iii) photocrosslinking reagents, and are composed of some or all of the following ligands (Scheme 1): Photoprobes (P), thermal probes (T), label (L), and a linker which may contain a cleavable ligand (X). (i) Photolabeling reagents are used to attach a label (a tag) on biological (macro)molecules and are usually composed of a photoprobe and a label, often connected via a linker, but the label may also be in the form of radio isotope substitution on the photoprobe. (ii) Photoaffinity labeling reagents, which are used to identify and analyze receptors on biological macromol ecules by photochemical attachment of a label to the tar get. They are as a rule modified receptor substrates, and P. E.Nielsen (ed.), Photochemical Probes in Biochemistry, 1-9. @ 1989 by Kluwer Academic Publishers. 2 for low molecular substrates, their design and syn-thesis are too varied to allow any generalization and shall not be dealt with here. When the substrate is large or macro molecular, reagents of a more general nature can be used to modify them. Such reagents contain as a rule one (or photoprobe, a thermal probe (or a second photoprobe), a linker and a label. ( iii) Photocrosslinking reagents are used to study interaction between two or more (macro )molecules. They usually contain one thermal probe and one photoprobe (or two photoprobes) bound together by a linker, which may be cleavable. Furthermore, they often are required to be supplied with a label. The present paper contains a general discussion of the design and utility of reagents for photolabeling, photoaffinit y labeling, and photocrosslinking, wi th par ticular emphasis on the authors's own work in this area, i.e., with protein-DNA photoaffinity labeling and photo crosslinking, protein-protein photocrosslinking, and DNA photolabeling reagents. 1. Reagent design A priori, only the shortcomings of the synthetic chemist limit the design of reagents, and we will undoubtedly see many elegant and ingenious designs in the future. In principle, the synthetic chemist is asked to com bine a certain number of ligands in such a way that a certain number of chemical events can be timed in the resulting reagent (e.g., Scheme 1). Furthermore, the re- @ Photo Thermal ~ probe + probe + abel + IVV\JV\J + ___ IPI In ILl (Link." (CleaYable) Group Scheme 1 agent must be sufficiently soluble in water, and the pho toprobe must still be activable with light of a suitable wavelength (X » 300 nm is preferable, but it is not necessarily prohibitive to use light of a shorter wave length in some cases). Most important, the efficiency of the reagents must be good: but what is efficiency?