Affinity Modification Of Biopolymers

278 Pages·2017·14.077 MB·English

by  Knorre, Dmitri G

Affinity
Modification
of
Biopolymers
Authors
Dmitri G. Knorre
Valentin V. Vlassov
Institute of Bioorganic Chemistry
U.S.S.R. Academy of Sciences
Novosibirsk, Soviet Union
CRC Press
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Library of Congress Cataloging-in-Publication Data
Knorre, D. G. (Dmitri Georgievich)
Affinity modification of biopolymers.
Bibliography: p.
Includes index.
1. Affinity labeling. 2. Biopolymers—Affinity
labeling. 3. Proteins—Analysis. 4. Nucleic acids—
Analysis. I. Vlassov, Valentin Viktorovich. II. Title.
QP519.9.A37K66 1988 574.19’285 88-2883
ISBN 0-8493-6925-8
A Library of Congress record exists under LC control number: 88002883
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PREFACE
The fundamental property of proteins and nucleic acids is the ability to bind definite low
molecular weight ligands or other biopolymers. This phenomenon is usually called recog-
nition. It is the basis of a number of the most important biological events. Thus, self-
assembly of complicated structures such as ribosomes, chromatins, and viruses proceeds
due to mutual recognition of nucleic acids and proteins constituting these structures. The
first step of all numerous enzymatic reactions is the specific binding of substrates to the
active center of the respective enzyme. Hormones and neurotransmitters interact with specific
receptors of cell membranes giving rise to a definite biological response of the target cell.
Recognition of antigens by antibodies or receptors of T lymphocytes is one of the crucial
steps of the action of the immune system. The specific interaction of nucleic acid strands
with complementary sequences of nucleotides is the foundation of the mechanism of storage,
multiplication, and expression of the genetic information. This incomplete list of phenomena
concerning the formation of biological structures, cell metabolism, regulation, nerve impulse
transmission, heredity, and immune response clearly demonstrates the extreme significance
of recognition processes in biological systems.
Recognition is not usually followed by any changes of the chemical structure of a bio-
polymer. Thus, enzymatic conversion of substrates is not accompanied by any alteration of
the chemical structure of the enzyme if transient covalent binding of certain fragments of
substrates to enzymes in the course of formation of labile intermediates is not taken into
account. This permits the repeated use of the same biopolymer molecule. At the same time,
the absence of chemical consequences of recognition makes it difficult to determine the area
of biopolymer participating in the specific complex formation.
Modern biochemistry demonstrates that it is usually possible to introduce changes in the
ligand structure which do not severely damage the ability of the structure to be recognized
by the respective biopolymer. This means that in the modified ligand the points are touched
which are not critically essential for the specific complex formation. In particular, some
reactive groups may be inserted into the ligand. In this case, the specific complex formation
may result in the chemical reaction between this reactive group and biopolymer residue
adjacent to the recognition area. This reaction should strongly predominate over reactions
of the same compound with similar residues of biopolymer due to close contact between
reaction partners. Therefore, selective chemical modification of a definite area of biopolymer
close to the region of specific interaction may be performed. As the selectivity is achieved
due to affinity of the reactive ligand, the process may be called affinity modification of
biopolymers.
As a general approach to a specific attack of enzymes and other functional proteins,
affinity modification was first realized by B. R. Baker.' As the region where recognition
and subsequent catalytic transformation of a substrate proceeds is usually referred to as
active site (active center), the approach as well as his book were called Design of Active
Site Directed Irreversible Inhibitors.
About the same time, N. I. Grineva started to elaborate the similar approach to selective
modification of nucleic acids. It was proposed to attach reactive groups to oligonucleotides
with sequences complementary to a definite region of the target nucleic acid. The method
was called complementary addressed modification.2•3 Oligonucleotide moiety of such re-
agents may be considered as an address directing the reactive group to the desired fragment
of nucleic acid subjected to modification.
In the 2 following decades affinity modification was widely used to study enzymes,
receptors, immunoglobulins, nucleoproteins, and other biopolymers and complexes. To more
easily localize the residues introduced into biopolymers by means of affinity modification,
reagents were usually supplied with radioactive labels. Therefore, the method is referred to
as affinity labeling. It has become one of the powerful tools of investigating structure and
function of proteins, nucleic acids, and complexes of proteins and nucleic acids. Pharma-
cological applications of the approach were discussed in a set of papers. Different aspects
of affinity labeling were considered in some reviews. A special volume of Methods in
Enzymology' was devoted to this method. In 1981, the Federation of the European Bio-
chemical Society (FEBS) organized special practical courses for affinity labeling in the
Institute of Organic Chemistry in Novosibirsk, U.S.S.R. The lectures given by leading
scientists in the field were edited as a separate book.'
The goal of this book is to give a systematic description of the main principles of affinity
modification and applications, consideration of possibilities, and restrictions of the method.
Modification within specific complexes is a special case of chemical modification which is
widely used in the nonaddressed version in biochemistry and related areas. Therefore, we
have included in the first introductory chapter of the book general considerations of chemical
modifications of biopolymers and the application of biopolymers.
The main principles and possibilities of affinity modification are illustrated by a great
number of experimental works covering the fields of biochemistry, molecular biology,
pharmacology, and immunochemistry. Therefore, the primary knowledge of the main con-
cepts of these disciplines by the readers is suggested. However, short descriptions of bio-
and immunochemical systems considered in the experimental examples are given in the text.
Among numerous types of specific interactions, the recognition of nucleic acids and the
components by proteins attracts the greatest attention of scientists. Several hundred enzymes
deal with nucleotides, nucleosidedi-, and triphosphates, and nucleotide coenzymes as sub-
strates and effectors. Almost all proteins and nucleoproteides participating in the storage,
multiplication, and expression of genetic information interact specifically with nucleic acids.
Among them are DNA and RNA polymerases, ribosomes, translation and transcription
factors, enzymes of the reparation and recombination systems, different DNA unwinding
proteins, and aminoacyl-tRNA-synthetases. Therefore, some preference is given in this book
to affinity labeling of biopolymers with reactive derivatives of nucleic acids and components.
A special section is devoted to this group of compounds and the results obtained by affinity
modification of polymerases of nucleic acids, ribisomes, and aminoacyl-tRNA-synthetases
are given in more detail as compared with the numerous number of other systems discussed
in this book.
It is impossible to represent the complete survey of the several thousand scientific papers
dealing with affinity modification. At the same time, we tried to present all the reviews
concerning the problems and achievements of the last few years.
THE AUTHORS
Dmitri G. Knorre, Professor, is the Director of the Novosibirsk Institute of Bioorganic
Chemistry of the Siberian Division of the U.S.S.R. Academy of Sciences. He was a student
of Mendeleev Institute of Technology in Moscow. As a postgraduate student, he worked at
the Institute of Chemical Physics, and in 1951 he was awarded the doctor's degree in
chemistry. In 1968, he became a correspondent member and, in 1981, full member of the
U.S.S.R. Academy of Sciences.
Professor Knorre's interest in biochemistry dates back to 1956 when, still in Moscow, he
started the first exercises in bioorganic chemistry of peptides and then in nucleic acids
chemistry. In 1961, he moved to the Institute of Organic Chemistry in Novosibirsk, and,
until 1984, headed the Department of Biochemistry in this institute. Since 1984, Professor
Knorre has been the Director of a newly formed Institute of Bioorganic Chemistry in
Novosibirsk.
His scientific interests were concentrated around chemical modification of biopolymers,
mainly affinity modification of nucleic acids with reactive derivatives of complementary
oligonucleotides (complementary addressed modification of nucleic acids). His main achieve-
ments are the elucidation of a mechanism of phosphorylation including some steps of ol-
igonucleotide synthesis, design of a series of new nucleoside triphosphate derivatives, iden-
tification of ribosomal proteins involved in interaction with transfer and messenger RNAs
by means of affinity modification with reactive derivatives of oligo- and polynucleotides.
Being the Head of the Chair of Molecular Biology at the Novosibirsk State University,
Professor Knorre holds lectures in physical chemistry, molecular biology, bioorganic chem-
istry, and biochemistry.
Valentin V. Vlassov is the Deputy Director of the Novosibirsk Institute of Bioorganic
Chemistry. Being a student of the Novosibirsk State University, he started experimental
work in Prof. D. G. Knorre's laboratory in the Institute of Organic Chemistry in 1967. In
1972, he received a doctor's degree. Since 1982, Dr. Vlassov has been a head of the
Laboratory of Biochemistry of Nucleic Acids, studying biochemical and biological appli-
cations of oligonucleotide derivatives.
A basic interest of Dr. Vlassov concerns the chemical modification of biopolymers with
special regard to specific modifications of nucleic acids. His main contributions to this field
have been to develop new approaches for the investigation of tertiary structure and molecular
interactions of RNAs by means of chemical modification with alkylating reagents, nitrogen
mustard, and ethyl nitrosourea, to design new derivatives of oligo- and polynucleotides, and
to study the possibility of using reactive oligonucleotide derivatives for sequence-specific
modification of DNA, specific arrest of mRNA translation, and suppression of viruses
multiplication. He holds lectures on chemistry of biopolymers at the Novosibirsk State
University.
TABLE OF CONTENTS
Chapter 1
Molecular Recognition and Chemical Modification of Biopolymers — Two Main Com-
ponents of Affinity Modification
I.(cid:9) Molecular Recognition (cid:9) 1
A. Noncovalent Interactions and the Role in Molecular
Recognition (cid:9) 1
B. Intramolecular Recognition in Biopolymers and the Role in the
Formation of the Secondary and Tertiary Structure (cid:9) 6
C. Recognition in the Multisubunit Structure Formation:
Multipeptide Complexes, Double-Stranded Nucleic Acids,
and Nucleoproteins (cid:9) 10
D. Active Centers of Enzymes: Recognition of Substrates,
Effectors, and Templates (cid:9) 14
E. Recognition in the Regulatory Processes (cid:9) 15
F. Recognition Processes at the Cell Surface (cid:9) 16
G. Recognition in the Immune System (cid:9) 19
II.(cid:9) Chemical Modification of Biopolymers (cid:9) 21
A. Derivatives of Biopolymers (cid:9) 23
B. Chemical Modification and Sequencing of Biopolymers (cid:9) 25
C. Mapping of Secondary and Tertiary Structure of Biopolymers
by Chemical Modification (cid:9) 28
D. The Study of the Structure of Multisubunit Proteins and
Complexes of Biopolymers (cid:9) 32
E. Bifunctional Reagents in the Study of the Structure and
Complexes of Biopolymers (cid:9) 35
F. Chemical Modification in the Study of the Structure-Function
Relations of Biopolymers (cid:9) 38
Chapter 2
Affinity Modification — Organic Chemistry
I.(cid:9) Reactive Groups of Biopolymers (cid:9) 43
II. Reactive Groups of Affinity Reagents 45
A. Alkylating and Arylating Groups 45
B. Acylating, Sulfonylating, and Phosphorylating Groups 49
C. Carbonylic Compounds (cid:9) 51
D. Sulfhydryl and Disulfide Groups (cid:9) 53
E. Photoreactive Groups (cid:9) 54
F. Direct Photocross-Linking of Biopolymers with Specific
Ligands (cid:9) 60
G. Suicide Reagents (cid:9) 64
H. Metal-Containing Groups (cid:9) 75
III.(cid:9) Design of Affinity Reagents (cid:9) 80
A. Targeting (Addressing) Structures of Affinity Reagents (cid:9) 80
B. General Approaches to the Synthesis of Affinity Reagents (cid:9) 87
C. Reactive Derivatives of Biopolymers (cid:9) 92
D. Reactive Derivatives of Nucleic Acids and Nucleic Acid
Components (cid:9) 96
Chapter 3
Affinity Modification — Experimental Methods
I.(cid:9) Introduction (cid:9) 109
II.(cid:9) Performance of Affinity Modification (cid:9) 109
A. General Procedures (cid:9) 109
B. Photoaffinity Modification (cid:9) 111
III.(cid:9) Experimental Procedures Used for Quantitative Investigation of
Modification of Biopolymers (cid:9) 113
A. Extent of Modification (cid:9) 113
B. Inactivation of Biopolymer (cid:9) 116
IV.(cid:9) Determination of the Modification Points (cid:9) 118
A. Distribution of the Label Among Subunits in Multisubunit
Systems (cid:9) 118
B. Determination of the Monomer Residues Modified (cid:9) 120
Chapter 4
Affinity Modification — Physical Chemistry
I.(cid:9) Kinetics of Affinity Modification (cid:9) 129
A. The Simplest Scheme of Affinity Modification and the Range
of Validity (cid:9) 129
B. Simultaneous Enzymatic and Affinity Modification Processes (cid:9)134
C. Simultaneous Modification of Several Independent Centers by
the Same Reagent (cid:9) 136
D. Main Sources of Kinetic Complications in Affinity
Modification (cid:9) 139
E. Affinity Modification with Unstable Reagents (cid:9) 142
F. Kinetics of Suicide Inhibition (cid:9) 145
G. Cooperative Effects in Affinity Modification (cid:9) 148
H. General Rules for Kinetic Treatment of Complicated Affinity
Modification Processes (cid:9) 151
II.(cid:9) Quantitative Criteria of Affinity Modification (cid:9) 157
A. General Direct Criteria (cid:9) 157
B. Comparative Criteria of Affinity Modification (cid:9) 159
C. Dynamic Aspects of Affinity Modification (cid:9) 163
Chapter 5
Affinity Modification in Biochemistry, Biology, and Applied Sciences
I.(cid:9) Introduction (cid:9) 165
II.(cid:9) Biochemical Applications of Affinity Modification (cid:9) 165
A. Structural Studies of the Active Centers of Enzymes (cid:9) 165
B. Cooperative Interactions in Enzymes as Revealed by Affinity
Modification (cid:9) 170
C. Affinity Modification of the Nucleic Acid Polymerases (cid:9) 172
D. Affinity Modification as a Tool of the Study of Functional
Topography of Ribosomes (cid:9) 177
E. Complementary Addressed Modification of Nucleic Acids (cid:9) 183
III.(cid:9) Biological, Pharmacological, and Therapeutic Applications of Affinity
Modification (cid:9) 187
A. Localization of the Ligand Binding Sites (cid:9) 187
B. Affinity Modification of Enzymes in Biological Systems (cid:9) 188
C. Affinity Modification of Cell Receptors (cid:9) 190
D. Affinity Modification as an Approach to Produce Locally
Damaged Biopolymers (cid:9) 192
E. Pharmacological and Therapeutic Applications of Affinity
Modification (cid:9) 193
Appendix (cid:9) 197
References (cid:9) 225
Index (cid:9) 265
1
Chapter 1
MOLECULAR RECOGNITION AND CHEMICAL MODIFICATION OF
BIOPOLYMERS — TWO MAIN COMPONENTS OF AFFINITY MODIFICATION
I. MOLECULAR RECOGNITION
A. Noncovalent Interactions and the Role in Molecular Recognition
Recognition results in the formation of a specific complex of biopolymers and ligands.
If the stability of a complex with a definite ligand significantly exceeds that of complexes
with similar compounds we refer to the phenomenon as recognition of this ligand by the
polymer under consideration.
Complexes are formed by noncovalent interactions of some groups of biopolymers with
respective groups of ligands. Three main types of interactions participate in recognition:
electrostatic, hydrogen bonding, and hydrophobic interactions.
Electrostatic interactions operate between groups bearing opposite charges. Amino acids
bear a positive charge at the amino groups in a protonated state and a negative charge at
the carboxylic group in an ionized state. However, the formation of a peptide bond results
in the disappearance of these charges and the main polypeptide chain of protiens is electro-
neutral with N-terminal NI-11- and C-terminal COO- residues.
NH3-CH-CO (cid:9) NH-CH-CO-NH-CH-CO (cid:9) NH-CH-000
(I)
R1(cid:9) Ri(cid:9) Ri.„1(cid:9) RN
Therefore, the main carriers of electric charges in proteins are side-chain groups. These
groups are presented in Table 1.
On the contrary, the main chain of nucleic acids
0 -P-0 Basel
5-end
R
,P
0 ‘0 Basel
Base i+1
R
R=H DNA(cid:9) 0' \0 ~Basen
R=OH RNA
OH R
3-e nd
is a carrier of negative charge and, therefore, all nucleic acids are polyanions.
Hydrogen bonding occurs between the polar X-H group (mainly X = N or 0) and electron
pairs of nonbonding orbitals of F, 0, or N atoms. In hydrogen bonding, one of the bonded
atoms is the donor, and the other is an acceptor of the proton. The peptide bond contains
donor N-H and acceptor CC0 groups. Many amino acids (Table 1) bear hydrophilic residues