Table Of ContentPhotochemical Probes in Biochemistry
NATO ASI Series
Advanced Science Institutes Series
A Series presenting the results of activities sponsored by the NA TO Science Committee,
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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
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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?
