Methods in Protein Sequence Analysis

580 Pages·1982·30.492 MB·English

by  Russell F. Doolittle (auth.), Marshall Elzinga (eds.)

Methods in Protein Sequence Analysis
Experimental Biology and Medicine
Methods in Protein Sequence Analysis, edited by
Marshall Elzinga, 1982
Inflammatory Diseases and Copper, edited by John R. J.
Sorenson, 1982
Membrane Fluidity: Biophysical Techniques and Cellular
Regulation, edited by Morris Kates and Arnis Kuksis,
1980
Methods in
Protein Sequence
Analysis
Edited by
Marshall Elzinga
Brookhaven National Laboratory, Upton, New York
Humana Press· Clifton, New Jersey
Organization and Program Committee:
Marshall Elzinga, Chairman
Lowell H. Ericsson
Agnes Henschen
Marcus J. Horn
Richard A. Laursen
John H. Walker
The organizers are grateful to the following for providing finan
cial support for this Conference:
National Science Foundation
AAA Laboratory
Beckman Instruments
Dionex Corporation
Hewlett-Packard Company
MCB Manufacturing Chemists
Pierce Chemical Company
Sequemat/Genetic Design, Inc.
Waters Associates, Inc.
Library of Congress Cataloging in Publication Data
Main entry under title:
Methods in protein sequence analysis.
(Experimental biology and medicine)
"Augmented record of the IVth International Conference
on Methods in Protein Sequence Analysis held September
25-25, 1981 at the Brookhaven National Laboratory,
Upton, New York"-
Includes index.
1. Amino acid sequence-Congresses. 2. Proteins-
Analysis-Congresses. I. Elzinga, Marshall. II. Inter
national Conference on Methods in Protein Sequence
Analysis (4th: 1981: Brookhaven National Laboratory)
III. Series: Experimental biology and medicine (Humana
Press) [DNLM: 1. Proteins-Analysis. 2. Amino acid
sequence. QU 60 M592]
QP551.M393 574.19'245 82-80733
ISBN-13: 978-1-4612-5834-6 e-ISBN-13: 978-1-4612-5832-2
001: 10.1007/978-1-4612-5832-2
©7982 The HUMANA Press Inc.
Softcover reprint of the hardcover 1st edition 1982
Crescent Manor
PO Box 2748
Clifton, NJ 07075
All rights reserved
No part of this book may be reproduced, stored in a retrieval system,
or transmitted, in any form or by any means, electronic, mechanical,
photocopying, microfilming, recording, or otherwise without written
permission from the Publisher.
PREFACE
Methods in Protein Sequence Analysis contains an intensely prac
tical account of all the new methodology available to scientists
carrying out protein and peptide sequencing studies. Many of the
striking advances in fields as diverse as immunology, cell motility,
and neurochemistry have in fact been fueled by our ever more
powerful ability to determine the sequences and structures of key
proteins and peptides. It is our hope that the rich array of tech
niques and methods for sequencing proteins discussed in this
volume-methods that generate much of the information crucial
to progress in modern biology-will now become accessible to
all who can benefit from them.
The papers of the present volume constitute the Proceedings of
the IVth International Conference on Methods in Protein Se
quence Analysis, which was held at Brookhaven National Labo
ratory, Upton, NY, September 21-25, 1981. It was the most recent
in a series of biennial conferences, the previous one having been
held in Heidelberg, GFR, in 1979. The series was originated by
Richard Laursen, and initially dealt with one aspect of the field,
solid-phase sequencing.
The scope of the meeting was very broad and among the many
aspects of protein sequencing discussed were: instrumentation,
strategy, chemicals, mass spectrometry, cleavage of proteins and
separation of peptides, and solid, liquid, manual, and even "gas
phase" sequencing.
The goals of the methods described here are essentially the
same as they were 30 years ago: one wants to know the order of
the amino acids in a given protein or peptide because the amino
acid sequence is information that is fundamental to understand
ing its properties. Thus many of the papers describe improve
ments of the traditional methods used to determine the complete
sequences of pure proteins. Especially notable in this regard are
the important recent developments that have taken place in the
area of peptide separation: here HPLC (high performance liquid
chromatography) is rapidly replacing all other column and paper
methods.
v
vi Preface
Similarly, the enormous advances being made in the technol
ogy of RNA and DNA sequencing have forced a re-evaluation of
many studies that involve protein sequencing. It is now clear that
protein and nucleic acid sequencing are complementary tech
niques, each with advantages in addressing different aspects of
many questions about structures and sequence. Thus, if one can
now prepare the right piece of nucleic acid, information regard
ing the amino acid sequence of the protein(s) for which it codes
can often be obtained very quickly by sequencing the nucleic
acid. However, questions regarding gene expression, posttrans
lational processing, sidechain modification, and so on generally
still require protein sequence data. In modern molecular biology,
crucial experiments sometimes require limited amounts of se
quence information from specific parts of prateins that are avail
able in only extremely small quantities. The design of such experi
ments and the development of the necessary micromethods for
sequencing at picomole levels are discussed in several of the
papers.
The scientific program of the conference included both oral
and poster presentations. In general, papers dealing primarily
with methods were presented orally and appear in this volume as
full manuscripts, while new sequences were presented as posters
and are published as "Communications." Most of the papers pro
vide up-to-date accounts of the authors' experiences in the devel
opment of procedures or the improvement of techniques. This
book should therefore be useful to practitioners of the protein
sequencing art who want to learn about new procedures and al
ternative methods that are potentially applicable to their own
sequencing problems, as well as to those who work in peripheral
areas and wish to familiarize themselves with developments in
this field.
April, 1982 Marshall Elzinga
Memorial
Erhard Gross, J9 28- J9 8 J,
In Memoriam
Christian Birr
Max-Planck-Institut fur Medizinische Forschung, Jahnstrasse 29,
Heidelberg, F. R. Germany
The international natural sciences community has lost one of its
most active, eager, busy and great members. On the 12th of Sep
tember, 1981, my friend and mentor, Dr. Erhard Gross, died in a
tragic traffic accident in Germany, ten days after the 53rd ann
iversary of his birthday. The cruelty of fate has thus taken him
from his wonderful wife, Gertrud, and his gentle and talented
sons, Johannes and Christoph. I am privileged by the organizer of
vii
viii Memorial
this symposium to briefly commemorate the academic life of Dr.
Erhard Gross.
He graduated in chemistry 1956 from the University of
Frankfurt, F. R. Germany, at the Institute of Organic Chemistry
headed by Theodor Wieland. In those early days Erhard Gross
developed a close relationship with his scientific teacher and
friendly mentor. This relationship matured into a trustful friend
ship with Theodor Wieland, which spanned Erhard's too short life.
It was a characteristic of Erhard to infuse his fellow-scholars, as
well as later students of Theodor Wieland, with his warm feelings
for his teacher. In this way, I received the honor and pleasure of
Dr. Gross' constant friendship and helpful advice.
In 1958 he passed the Ph.D. examinations with the presentation
of his thesis on the synthesis of a bicyclic peptide similar in struc
ture to the mushroom toxin Phalloidin, which he had studied under
the auspices of Th. Wieland at the University of Frankfurt. As we
shall see, there are three common threads that connect the scien
tific efforts and results of the natural scientist and organic chemist
Dr. Gross: his biomedical interest, the peptide bond, and
thioethers, three characteristics of his doctoral thesis.
Just after his marriage in 1958 Erhard left Frankfurt, Germany,
for the United States of America. Actually, this was not his first ex
perience with the American people. In 1945 he became an Ameri
can Prisoner of War for a short period after having had to take
part as a youngster in the Second World War in 1944.
In 1958 Dr. Gross started to work in the prestigious chemistry
laboratory of B. Witkop at the Institute of Arthritis and Metabolic
Diseases of the National Institute of Health (NIH) in Bethesda,
Maryland. He was charged with the search for nonenzymatic
methods in the cleavage of peptides and proteins. It was during
this time that Erhard Gross developed the world-famous cyano
gen bromide method for the selective cleavage of methionine
peptide bonds, which was published in 1961 together with B.
Witkop. To date this specific reaction remains the most useful
chemical procedure in the arsenal of methods available to pro
tein sequencers. Without the specificity of the cyanogen bromide
cleavage we should not have the great successes in the industrial
biosynthesis in bacteria of peptide hormones by the recombinant
DNA technique. There, methionine is incorporated between the
carrier protein and the target peptide as a specific detachment
site.
In the mid sixties Dr. Gross investigated reaction mechanisms
of enzymes, and detected aspartic acid in the active center of
pepsin. He elucidated the structure of uncommon amino acids
Memorial ix
and discovered dehydro-alanine as a natural constituent in
proteins.
In 1968 Erhard Gross became Chief of the Molecular Structure
Section at the Laboratory of Biomedical Sciences of the National
Institute of Child Health and Human Development at the NIH. His
research in cell biology was concentrated on the isolation and
characterization of cell organelles. He investigated placental
Iysosomes and those from polymorphous leucocytes. His chem
ical work was centered on the structural elucidation and synthesis
of membrane active peptides for the induction of fetal resorption.
For this purpose he investigated the gramicidins and published in
1969-1971 the syntheses of Gramicidin A, C and B. His most re
cent research work was focused upon the conformational char
acteristics of microbial peptides that lead to formation of channel
aggregates and thus allow the translocation of cations across
lipid membranes.
In 1971 Erhard described the pentacyclic heterodetic nature of
the polypeptide Nisin, an antibiotic from Streptococcus lactis
which contains 5 thioether bridges. In 1972 he correlated these
structural elements with those of the other pentacyclic thioether
polypeptide Subtilin, a microbial product from Bacillus subtilis.
Since then a great deal of his research efforts were devoted to the
task of synthesizing those very difficult polycyclic thioether pep
tides. Though he and his coworkers succeeded in the preparation
of a monocyclic element of the antibiotics, by his untimely death
the total syntheses of Nisin and Subtilin have to remain a chal
lenge for Erhard's scientific heirs.
Erhard Gross often demonstrated his talents in scientific man
agement, which culminated in his brilliant organization of the
most successful Sixth American Peptide Symposium in 1979 at
Georgetown University in Washington, D.C. Together with his
friend and colleague Johannes Meienhofer, Erhard most effi
ciently edited, in an intellectual marathon, more than a thousand
pages of the proceedings volume, which was published within
less than six months. Most recently, the same team of authors and
editors received great international recognition and admiration
through the publication of their series "THE PEPTIDES, Analysis,
Synthesis, Biology." Three excellent volumes have already ap
peared and a fourth is in preparation. By these monuments of
work our memorial to Erhard Gross will remain immortal.
His scientific contributions were honored by the Humboldt
Award presented to him in 1978.
In my mind's eye I can see Erhard Gross with his famous trade
marks, the bow-tie, his red-blond crest and his finger pointing as
x Memorial
an impulsive discussant. His enthusiasm and activity would have
helped greatly to make a meeting like this one a success. Our best
memorial to him will be to remember these distinguishing charac
teristics, and use them as an inspiration for a happy and fruitful
symposium.